Kits for single-step analyte detection with process control

ABSTRACT

Kits for detecting analyte polynucleotides and an internal control in a sample. Included in the kit are an internal control polynucleotide and amplification reagents to co-amplify a first analyte polynucleotide and the internal control. Also included are first and second hybridization probes, each having a label indistinguishable from the other. The probes are respectively capable of hybridizing with a first analyte amplicon and an internal control amplicon. The first and second labels are indistinguishable homogeneous labels.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/657,749, filed Oct. 18, 2019, which is a continuation of U.S.application Ser. No. 15/729,128, filed Oct. 10, 2017, now U.S. Pat. No.10,487,353, which is a continuation of U.S. application Ser. No.13/173,558, filed Jun. 30, 2011, now U.S. Pat. No. 9,822,397, whichclaims the benefit of U.S. Provisional Application No. 61/360,296, filedJun. 30, 2010. The disclosures of these prior applications are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to the sub-field of biotechnology thatconcerns diagnostic assays. More particularly, the invention relates toanalyte detection in an assay that includes an internal control, wherethe analyte and internal control are detected using different probes. Ina highly preferred embodiment, nucleic acid analytes are detected in asingle mixture using different hybridization probes that harboridentical detectable labels.

BACKGROUND

Modern assays for detecting the presence of an analyte (e.g., a nucleicacid, protein, lipid, carbohydrate, and the like) rely on the use of apositive control to verify process reliability. For example, an assaymay seek to detect a target nucleic acid using nucleic acidamplification followed by probe hybridization and detection. The sampleundergoing amplification can include an internal control (hereafter,“IC”) nucleic acid that co-amplifies with the analyte nucleic acid.Amplification products advantageously can have non-identical sequencesthat can be detected using different hybridization probes. Detection ofthe IC amplification product verifies integrity of the amplification anddetection components of the assay procedure. That information is usefulwhen there is a failure to detect analyte amplification products.Detection of the IC signal, in such a case, validates theanalyte-negative result. Hybridization probes specific for analyteamplicons, and for IC amplicons are conventionally distinguished eitherby the labels they harbor, or by spatial separation.

Probe-based assays, including protein and nucleic acid assays, thatinclude an internal process control commonly make one of the followingdistinctions with respect to detection of IC and analyte: (1) IC isdetected separate from analyte; and (2) analyte is detected separatefrom the combination of analyte plus IC. U.S. Pat. No. 6,586,234illustrates both of these possibilities using two-read systems fordetection of IC and analyte nucleic acids. When analyte nucleic acid isdetected independent of the combination of IC plus analyte, the latterhybridization signal can be evaluated for samples yielding analytehybridization signals that fall below a threshold cutoff required forpositive scoring. For example, a signal below the threshold cutoff foranalyte detection may alternatively indicate absence of analyte, ormalfunction in the assay. If a signal is detected in a second read thatrepresents the combination of IC plus analyte, that result isinterpreted as validating the analyte-negative result. In other words,detection of an adequate signal for IC plus analyte indicates that ICmust have been detected, and so can validate an analyte-negative result.It should be apparent that success of such a system depends on theability to separate the analyte hybridization signal from thecombination of hybridization signals representing IC and analyte.

One difficulty encountered in the field of analyte detection concernsthe number of different labels required for analysis of multiplexreactions when detection is carried out without spatial separationbetween different probes (e.g., the different probes being in fluidcommunication, and free in solution rather than immobilized). This maybe understood in the context of an assay that co-amplifies an IC nucleicacid and two different target nucleic acids. With the IC probe harboringone label, a collection of probes for detecting the remaining twotargets can be labeled with a second label. A positive detection signalfor the second label indicates that one of the two analytes is present,but fails to differentiate one from the other. As the number of analytesclimbs, the amount of re-testing needed to resolve the reactive speciesincreases similarly. Stated differently, the burden of re-testing toidentify the reactive species in positively scoring multiplex assays isa disadvantage, especially when the fraction of positive samples becomessignificant.

The present invention addresses the need for simplified analyteidentification systems.

SUMMARY

In one aspect the invention relates to an apparatus for determining,with process control, the presence or absence of a first analyte in asample that includes an internal control. Generally speaking, theinvented apparatus includes: (a) a holder configured to contain thesample; (b) an optical detection mechanism arranged to receive opticalsignals from the sample when contained in the holder; and (c) aprocessor (e.g., a computer) in communication with the optical detectionmechanism, the processor being programmed to perform the step ofdetermining which of a number of situations applies. In accordance withthe invention, in addition to the internal control the sample furtherincludes: an internal control probe that generates an internal controlsignal after contacting the internal control; a first analyte probe thatgenerates a first analyte signal after contacting the first analyte, ifpresent in the sample; and optionally a second analyte probe thatgenerates a second analyte signal after contacting a second analyte, ifpresent in the sample. Further in accordance with the invention, theoptical detection mechanism is configured to measure a combined signalgenerated by the internal control probe and the analyte probe withoutdistinguishing the internal control signal from the analyte signal. Theoptical detection mechanism is optionally configured to measure thesecond analyte signal generated by the second analyte probe. Stillfurther in accordance with the invention, the processor is programmed todetermine which of the following situations applies: (i) the sample doesnot include the first analyte if the magnitude of the combined signal isless than a first analyte cutoff value and either (1) the magnitude ofthe combined signal is greater than or equal to a validity cutoff value,or (2) the second analyte probe is included in the sample, the opticaldetection mechanism is configured to measure the second analyte signal,and the magnitude of the second analyte signal is greater than or equalto a second analyte cutoff value, thereby establishing that the sampleincludes the second analyte; (ii) the sample includes the first analyteif the magnitude of the combined signal is greater than or equal to thefirst analyte cutoff value, and (iii) it cannot be determined whether ornot the sample includes the first analyte if the magnitude of thecombined signal is less than the first analyte cutoff value and lessthan the validity cutoff value, and, if the second analyte probe isincluded in the reaction mixture, and the optical detection mechanism isconfigured to measure the second analyte signal, the second analytesignal is less than the second analyte cutoff value. Generally, thefirst analyte cutoff value is a signal amount greater than the validitycutoff value, and the detectable maximum of the internal control signalcannot exceed the first analyte cutoff value.

In accordance with a first highly preferred embodiment of the generallydescribed apparatus for determining, with process control, the presenceor absence of a first analyte in a sample that includes an internalcontrol, the optical detection mechanism is configured to measure thesecond analyte signal generated by the second analyte probe. When thisis the case, it is preferred that the holder does not substantiallychange temperature during operation of the optical detection mechanismto measure the combined signal; the first analyte, the second analyte,and the internal control each include nucleic acid; and the opticaldetection mechanism does not measure the first analyte signal withoutalso measuring the internal control signal. Alternatively, it ispreferred that the optical detection mechanism does not measure thefirst analyte signal without also measuring the internal control signal;and that the apparatus further includes an output device that produces atangible record (e.g., a printed record, or an electronic record storedon computer-readable media) of the determining step performed by theprocessor. More preferably, the holder does not substantially changetemperature during operation of the optical detection mechanism tomeasure the combined signal; the first analyte, the second analyte, andthe internal control each include nucleic acid; and the opticaldetection mechanism does not measure the first analyte signal withoutalso measuring the internal control signal. When this is the case, it ispreferred that the sample maintains substantially constant temperatureduring operation of the optical detection mechanism to measure thecombined signal. This can, for example, involve the use of atemperature-controlled incubator. Alternatively, it is preferred thatthe optical detection mechanism includes a detector selected from thegroup consisting of a luminometer and a fluorometer. More preferably,the detector is the luminometer.

In accordance with a second highly preferred embodiment of the generallydescribed apparatus for determining, with process control, the presenceor absence of a first analyte in a sample that includes an internalcontrol, the optical detection mechanism is not configured to measurethe second analyte signal generated by the second analyte probe. Whenthis is the case, it is preferred that the holder does not substantiallychange temperature during operation of the optical detection mechanismto measure the combined signal; the first analyte and the internalcontrol each include nucleic acid; and the optical detection mechanismdoes not measure the first analyte signal without also measuring theinternal control signal. Alternatively, the optical detection mechanismdoes not measure the first analyte signal without also measuring theinternal control signal, and the apparatus further includes an outputdevice that produces a tangible record of the determining step performedby the processor. More preferably, the holder does not substantiallychange temperature during operation of the optical detection mechanismto measure the combined signal; the first analyte and the internalcontrol each include nucleic acid; and the optical detection mechanismdoes not measure the first analyte signal without also measuring theinternal control signal. When this is the case, it is preferred that thesample maintains substantially constant temperature during operation ofthe optical detection mechanism to measure the combined signal.Alternatively, it is preferred that the optical detection mechanismincludes a detector selected from the group consisting of a luminometerand a fluorometer. More preferably, the detector is the luminometer.

In addition to the foregoing highly preferred embodiments of thegenerally described apparatus, there also are a number of generallypreferred variations that can be used to modify the invented apparatus.In one generally preferred embodiment, the sample is contained in areaction receptacle, and the holder is configured to contain a pluralityof reaction receptacles. More preferably, the reaction receptacle isselected from the group consisting of a tube, and a well of a multiwellplate. In another generally preferred embodiment, the optical detectionmechanism includes a detector selected from the group consisting of aluminometer and a fluorometer. More preferably, the detector is theluminometer. In still another generally preferred embodiment, theprocessor is a computer (e.g., such as a stand-alone computer) thatincludes a software look-up table. In yet another generally preferredembodiment, the holder does not substantially change temperature duringoperation of the optical detection mechanism to measure the combinedsignal; the first analyte, the second analyte, and the internal controleach include nucleic acid; and the optical detection mechanism does notmeasure the first analyte signal without also measuring the internalcontrol signal. In still yet another generally preferred embodiment, thesample maintains substantially constant temperature during operation ofthe optical detection mechanism to measure the combined signal. Morepreferably, wherein the holder is contained within atemperature-controlled incubator. In still yet another generallypreferred embodiment, the optical detection mechanism does not measurethe first analyte signal without also measuring the internal controlsignal, and the apparatus further includes an output device thatproduces a tangible record of the determining step performed by theprocessor.

In another aspect, the invention relates to a method, employing processcontrol, for determining the presence or absence of a first analyte in asample that includes an internal control. Generally speaking, the methodincludes a first step (a) for preparing a reaction mixture to be testedfor the presence of the first analyte. The reaction mixture includes:the sample; an internal control probe that generates an internal controlsignal after contacting the internal control; a first analyte probe thatgenerates a first analyte signal after contacting the first analyte, ifpresent in the sample; and optionally a second analyte probe thatgenerates a second analyte signal after contacting a second analyte, ifpresent in the sample. Next, there is a step (b) for measuring: (i) acombined signal generated by the internal control probe and the analyteprobe without distinguishing the internal control signal from theanalyte signal; and (ii) optionally the second analyte signal generatedby the second analyte probe, if included in the reaction mixture. Next,there is a step (c) for determining which of the following situationsapplies: (i) the sample does not include the first analyte if themagnitude of the combined signal is less than a first analyte cutoffvalue, and either (1) the magnitude of the combined signal is greaterthan or equal to a validity cutoff value, or (2) the second analyteprobe is included in the reaction mixture, the second analyte signal ismeasured in step (b), and the magnitude of the second analyte signalmeasured in step (b) is greater than or equal to a second analyte cutoffvalue, thereby establishing that the sample includes the second analyte;(ii) the sample includes the first analyte if the magnitude of thecombined signal is greater than or equal to the first analyte cutoffvalue; and (iii) it cannot be determined whether or not the sampleincludes the first analyte if the magnitude of the combined signal isless than the validity cutoff value, and, if the second analyte probe isincluded in the reaction mixture, the second analyte signal is measuredin step (b), and the magnitude of the second analyte signal measured instep (b) is less than the second analyte cutoff value. Generally, thefirst analyte cutoff value is a signal amount greater than the validitycutoff value, and the detectable maximum of the internal control signalcannot exceed the first analyte cutoff value.

In accordance with a first highly preferred embodiment of the generallydescribed method, the reaction mixture prepared in step (a) includes thesecond analyte probe; step (b) includes measuring at a constanttemperature; and step (c) is automated by a computer. More preferably,step (b) includes measuring optically. In one instance, step (b)preferably includes measuring optically with a device selected from thegroup consisting of a luminometer and a fluorometer. More preferably,the device is the luminometer. In another instance, the internal controlprobe, the first analyte probe, and the second analyte probe are eachdetectably labeled. More preferably, the internal control probe and thefirst analyte probe are each detectably labeled with identicaldetectable labels. For example, the internal control probe and the firstanalyte probe are each detectably labeled with the same chemiluminescentlabel. Alternatively, the internal control probe and the first analyteprobe are each detectably labeled with the same acridinium ester.

In accordance with a second highly preferred embodiment of the generallydescribed method, the reaction mixture prepared in step (a) does notinclude the second analyte probe; step (b) includes measuring at aconstant temperature; and step (c) is automated by a computer. Morepreferably, step (b) includes measuring optically. In one instance, step(b) preferably includes measuring optically with a device selected fromthe group consisting of a luminometer and a fluorometer. Morepreferably, the device is the luminometer. In another instance, theinternal control probe, the first analyte probe, and the second analyteprobe are each detectably labeled. More preferably, the internal controlprobe and the first analyte probe are each detectably labeled withidentical detectable labels. For example, the internal control probe andthe first analyte probe are each detectably labeled with the samechemiluminescent label. Alternatively, the internal control probe andthe first analyte probe are each detectably labeled with the sameacridinium ester. In addition to the foregoing highly preferredembodiments of the generally described method, there also are a numberof generally preferred variations that can be used to modify theinvented method. In one generally preferred embodiment, step (b)includes measuring at a constant temperature, and step (c) is automatedby a computer. In another generally preferred embodiment, the reactionmixture prepared in step (a) includes the second analyte probe. Morepreferably, the second analyte signal is measured in step (b); themagnitude of the second analyte signal measured in step (b) is less thanthe second analyte cutoff value; and the magnitude of the combinedsignal measured in step (b) is greater than or equal to the validitycutoff value, thereby determining that the sample does not include thesecond analyte. In still another generally preferred embodiment, thereaction mixture prepared in step (a) does not include the secondanalyte probe. More preferably, the magnitude of the combined signalmeasured in step (b) is less than the first analyte cutoff value butgreater than or equal to the validity cutoff value, and it is determinedin step (c) that the sample does not include the first analyte. In yetanother generally preferred embodiment, each of the first analyte, theinternal control, and the second analyte include nucleic acid. In stillyet another generally preferred embodiment, the internal control probe,the first analyte probe, and the second analyte probe are eachdetectably labeled. More preferably, the internal control probe and thefirst analyte probe are each detectably labeled with identicaldetectable labels. When this is the case, it is preferred that theinternal control probe and the first analyte probe are each detectablylabeled with the same chemiluminescent label. Alternatively, itpreferred that the internal control probe and the first analyte probeare each detectably labeled with the same acridinium ester. In yet stillanother generally preferred embodiment, the internal control probe, thefirst analyte probe, and the second analyte probe each includechemiluminescent labels. In yet still another generally preferredembodiment, step (b) includes measuring optically. More preferably, step(b) includes measuring optically with a device selected from the groupconsisting of a luminometer and a fluorometer. Still more preferably,the device is the luminometer. In yet still another generally preferredembodiment, step (c) involves determining with a computer that includesa software look-up table. In yet still another generally preferredembodiment, each of the probes includes nucleic acid.

DETAILED DESCRIPTION

Introduction and Overview

The present invention provides tools and methods for identifyinganalyte-containing samples by detecting an IC signal and an analytesignal in the same reaction mixture using single channel detection,using only a single read, and without needing to distinguish signalscontributed by IC and analyte probe binding. In a highly preferredembodiment, a single detectable label species is used to label both theanalyte probe, as may be used for detecting analyte amplicons, and theIC probe, as may be used for detecting IC amplicons. In anotherembodiment, the labels can differ as long as they both can be detectedin a single channel of a detection device, and as long as the maximumsignal generated by IC detection probe falls below the threshold cutofffor analyte detection. In accordance with the described method, themagnitude of the signal representing detection of analyte and IC is usedas a variable, thus requiring a plurality of thresholds for interpretingresults. In this way it is possible to eliminate the requirement foreither spatial separation or separate labels to detect IC amplicons andanalyte amplicons, and to be able to validate a negative result foranalyte detection. This is particularly true when probe hybridization isassessed at constant temperature, as illustrated below, therebydistinguishing the disclosed technique from thermal melting analysis.Indeed, the described technique does not require monitoring the extentof probe hybridization under a plurality of temperature conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating how the magnitude of a combined ICsignal plus analyte signal is used in accordance with the disclosedtechnique to determine the presence or absence of analyte in anIC-validated assay. The vertical axis of the graph represents combinedsignal magnitude. Indicated on the vertical axis are validity (“A”) andanalyte (“B”) cutoffs that are used for interpreting experimentalresults. A combined signal falling below the validity cutoff cannotindicate valid assay results (i.e., possibly indicating a failed orinvalid reaction). A combined signal that meets or exceeds the validitycutoff, but does not meet or exceed the analyte cutoff indicates a validreaction that did not include analyte. A combined signal that meets orexceeds the analyte cutoff indicates a reaction that included analyte,and is automatically considered valid.

FIG. 2 is a bar graph showing results for a multiplex assay wherein twoanalytes were detected individually, or in combination. Open barsindicate the magnitude of the combined signal detected using probesspecific for IC and Analyte-1. These probes harbored the same type of AElabel (i.e., a flasher), and signals represent cumulative probehybridization signals in the homogenous assay format. All four trials(i.e., Neg control; Analyte-1 only; Analyte-2 only; and Analyte-1 &Analyte-2) gave detectable flasher signals. Filled bars indicate themagnitude of signal detected using a probe specific for Analyte-2, wherethat probe harbored a type of AE (i.e., a glower) different from thetype used for labeling the probes specific for the IC and Analyte-1.Only trials that included Analyte-2 nucleic acid templates yieldeddetectable Analyte-2 signals.

DEFINITIONS

The following terms have the indicated meanings in the specificationunless expressly indicated to have a different meaning.

By “sample” or “test sample” is meant any substance suspected ofcontaining a target organism or biological molecule, such as a nucleicacid derived from the target organism. The substance may be, forexample, an unprocessed clinical specimen, a buffered medium containingthe specimen, a medium containing the specimen and lytic agents forreleasing nucleic acid belonging to the target organism, or a mediumcontaining nucleic acid derived from the target organism which has beenisolated and/or purified in a reaction receptacle or on a reactionmaterial or device. In some instances, a sample or test sample maycomprise a product of a biological specimen, such as an amplifiednucleic acid to be detected.

By “analyte” is meant a substance, such as a nucleic acid or protein,that is detected or measured in an analytical procedure. The analyte maybe contained in a sample undergoing testing.

As used herein, “standard samples” are samples containing knownquantities of an analyte.

As used herein, “polynucleotide” means either RNA, DNA, or a chimericmolecule containing both RNA and DNA.

By “analyte polynucleotide” or “analyte nucleic acid” is meant apolynucleotide of interest that is to be detected or quantified.

By “analyte polynucleotide standard” is meant a known quantity of ananalyte polynucleotide, or fragment thereof. For example, a viralanalyte polynucleotide standard may contain a known number of copies ofa viral genome, viral transcript, or in vitro synthesized transcriptrepresenting a portion of the viral genome.

“Nucleic acid” refers to a multimeric compound comprising nucleosides ornucleoside analogs which have nitrogenous heterocyclic bases, or baseanalogs, which are linked by phosphodiester bonds or other linkages toform a polynucleotide. Nucleic acids include RNA, DNA, or chimericDNA-RNA polymers, and analogs thereof. A nucleic acid “backbone” may bemade up of a variety of linkages, including one or more ofsugar-phosphodiester linkages, peptide-nucleic acid (PNA) bonds (PCT No.WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, orcombinations thereof. Sugar moieties of the nucleic acid may be eitherribose or deoxyribose, or similar compounds having known substitutions,such as 2′ methoxy substitutions and 2′ halide substitutions (e.g.,2′-F). Nitrogenous bases may be conventional bases (A, G, C, T, U),analogs thereof (e.g., inosine), derivatives of purine or pyrimidinebases, such as N⁴-methyl deoxygaunosine, deaza- or aza-purines, deaza-or aza-pyrimidines, pyrimidine bases having substituent groups at the 5or 6 position, purine bases having an altered or replacement substituentat the 2, 6 and/or 8 position, such as 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine (U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” positions in whichthe backbone does not include a nitrogenous base for one or moreresidues (see U.S. Pat. No. 5,585,481). Nucleic acids also include“locked nucleic acids” (LNA), an analog containing one or more LNAnucleotide monomers with a bicyclic furanose unit locked in an RNAmimicking sugar conformation (Vester et al., 2004, Biochemistry43(42):13233-41). A nucleic acid may comprise only conventional sugars,bases, and linkages as found in RNA and DNA, or may include conventionalcomponents and substitutions (e.g., conventional bases linked by a 2′methoxy backbone, or a nucleic acid including a mixture of conventionalbases and one or more base analogs). Methods for synthesizing nucleicacids in vitro are well known in the art.

By “oligonucleotide” or “oligomer” is meant a polymer made up of two ormore nucleoside subunits or nucleobase subunits coupled together.Oligonucleotides preferably have a length in the range of from 10-100nucleotides, more preferably 10-80 nucleotides, and still morepreferably from 15-60 nucleotides. The oligonucleotide may be DNA and/orRNA and analogs thereof. The sugar groups of the nucleoside subunits maybe ribose, deoxyribose and analogs thereof, including, for example,ribonucleosides having a 2′-O-methylsubstitution to the ribofuranosylmoiety. Oligonucleotides including nucleoside subunits having 2′substitutions and which are useful as detection probes, capture oligosand/or amplification oligonucleotides are disclosed by Becker et al., inU.S. Pat. No. 6,130,038. The nucleoside subunits may be joined bylinkages such as phosphodiester linkages, modified linkages, or bynon-nucleotide moieties which do not prevent hybridization of theoligonucleotide to its complementary target nucleic acid sequence.Modified linkages include those linkages in which a standardphosphodiester linkage is replaced with a different linkage, such as aphosphorothioate linkage or a methylphosphonate linkage. The nucleobasesubunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo-peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine (DNA analogs having a pseudo-peptide backbone are commonlyreferred to as “peptide nucleic acids” or “PNA”, and are disclosed byNielsen et al., “Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082.) Othernon-limiting examples of oligonucleotides or oligomers contemplated bythe present invention include nucleic acid analogs containing bicyclicand tricyclic nucleoside and nucleotide analogs referred to as “LockedNucleic Acids,” “Locked Nucleoside Analogues” or “LNA.” (Locked NucleicAcids are disclosed by Wang, “Conformationally Locked Nucleosides andOligonucleotides,” U.S. Pat. No. 6,083,482; Imanishi et al.,“Bicyclonucleoside and Oligonucleotide Analogues,” U.S. Pat. No.6,268,490; and Wengel et al., “Oligonucleotide Analogues,” U.S. Pat. No.6,670,461.) Any nucleic acid analog is contemplated by the presentinvention, provided that the modified oligonucleotide can hybridize to atarget nucleic acid under either stringent hybridization conditions oramplification reaction conditions.

As used herein, “amplification” or “amplifying” refers to an in vitroprocedure for obtaining multiple copies of a target nucleic acidsequence, its complement or fragments thereof. For example, an in vitroamplification reaction is an enzyme-catalyzed reaction that results inthe synthesis of multiple copies of a target nucleic acid sequence, itscomplement or fragments thereof. Examples of amplification methods thatcan be used for preparing in vitro amplification reactions are givenbelow. Preferred in vitro amplification reactions synthesize ampliconsin an exponential fashion, meaning that one amplicon serves as thetemplate for production of new amplicons.

By “amplicon” or “amplification product” is meant a nucleic acidmolecule generated in a nucleic acid amplification reaction. An ampliconor amplification product contains a target nucleic acid sequence thatmay be of the same or opposite sense as the target nucleic acid.

By “analyte amplicon” or “analyte amplification product” is meant anamplicon synthesized using an analyte nucleic acid as the template in anucleic acid amplification reaction.

As used herein, “probe” refers to an analyte-specific reagent useful fordetection of a target, such as a target biological molecule. Examples ofprobes include nucleic acid hybridization probes, antibody probes, cellsurface receptors, and receptor-specific ligands.

By “hybridization” or “hybridize” is meant the ability of two completelyor partially complementary nucleic acid strands to come together underspecified hybridization assay conditions to form a stable structurehaving a double-stranded region. The two constituent strands of thisdouble-stranded structure, sometimes called a “hybrid,” are heldtogether by hydrogen bonds. Although these hydrogen bonds most commonlyform between nucleotides containing the bases adenine and thymine oruracil (A and T or U) or cytosine and guanine (C and G) on singlenucleic acid strands, base pairing can also form between bases which arenot members of these “canonical” pairs. Non-canonical base pairing iswell-known in the art.

As used herein, a “hybridization probe” is an oligonucleotide thathybridizes specifically to a target sequence in a nucleic acid,preferably in an amplified nucleic acid, under conditions that promotehybridization, to form a detectable hybrid. A probe optionally maycontain a detectable moiety which either may be attached to the end(s)of the probe or may be internal. The nucleotides of the probe whichcombine with the target polynucleotide need not be strictly contiguous,as may be the case with a detectable moiety internal to the sequence ofthe probe. Detection may either be direct (i.e., resulting from a probehybridizing directly to the target sequence or amplified nucleic acid)or indirect (i.e., resulting from a probe hybridizing to an intermediatemolecular structure that links the probe to the target sequence oramplified nucleic acid). The “target” of a probe generally refers to asequence contained within an amplified nucleic acid sequence whichhybridizes specifically to at least a portion of a probe oligonucleotideusing standard hydrogen bonding (i.e., base pairing). A probe maycomprise target-specific sequences and optionally other sequences thatare non-complementary to the target sequence that is to be detected.

As used herein, a “detectable label,” or simply “label,” is a chemicalmoiety that can be detected, or can lead to a detectable response.Detectable labels in accordance with the invention can be linked toprobes, such as hybridization probes, either directly or indirectly.Examples of preferred detectable labels include radioisotopes, enzymes,haptens, chromophores such as dyes or particles that impart a detectablecolor (e.g., latex beads or metal particles), luminescent compounds(e.g., bioluminescent, phosphorescent or chemiluminescent moieties) andfluorescent compounds.

As used herein, “homogeneous detectable label” refers to a label thatcan be detected in a homogeneous fashion by determining whether thelabel is on a probe bound to a target sequence. That is, homogeneousdetectable labels can be detected without physically removing bound(e.g., hybridized) from non-bound (e.g. unhybridized) forms of the labelor labeled probe. Homogeneous detectable labels are preferred when usinglabeled probes for detecting nucleic acids. Examples of homogeneouslabels have been described in detail by Arnold et al., U.S. Pat. No.5,283,174; Woodhead et al., U.S. Pat. No. 5,656,207; and Nelson et al.,U.S. Pat. No. 5,658,737. Preferred labels for use in homogenous assaysinclude chemiluminescent compounds (e.g., see Woodhead et al., U.S. Pat.No. 5,656,207; Nelson et al., U.S. Pat. No. 5,658,737; and Arnold, Jr.,et al., U.S. Pat. No. 5,639,604). Preferred chemiluminescent labels areacridinium ester (“AE”) compounds, such as standard AE or derivativesthereof (e.g., naphthyl-AE, ortho-AE, 1- or 3-methyl-AE,2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE,meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE,ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or3-methyl-meta-difluoro-AE, and 2-methyl-AE).

A “homogeneous assay” refers to a detection procedure that does notrequire physical separation of bound probe from non-bound probe prior todetermining the extent of specific probe binding. Exemplary homogeneousassays, such as those described herein, can employ molecular torches,molecular beacons or other self-reporting probes which have astem-and-loop structure and emit fluorescent signals when hybridized toan appropriate target, chemiluminescent acridinium ester labels whichcan be selectively destroyed by chemical means unless present in ahybrid duplex, and other homogeneously detectable labels that will befamiliar to those having an ordinary level of skill in the art.

In the context of the invention, certain methods are used for making oroutputting a diagnostic determination. For example, based on a set ofdata there will be a conclusion that the likelihood of a particularanalyte being present in a test sample is very high. An output result ofthe method can be indicated as a step for “determining” or “assigning”or “establishing” or “calling” that a particular analyte is present, orperhaps absent.

As used herein, an “internal control” is an agent included in a reactionmixture that is used for detecting the presence or absence of ananalyte, where detection of the internal control, directly orindirectly, serves to validate assay process steps. In the context of anassay that detects a nucleic acid analyte using an amplificationreaction, an internal control can be a nucleic acid template that can beco-amplified and detected in a hybridization reaction along with thenucleic acid analyte. Detection of internal control amplificationproducts at an appropriate level confirms success of the amplificationand hybridization process steps. In one embodiment, an internal controlnucleic acid amplifies using the same primers that amplify analytenucleic acid, but internal control amplicons and analyte amplicons aredetected using different hybridization probes. Preferred internalcontrols include exogenous agents that are added to reaction mixturesused for detecting the presence or absence of analytes.

By “internal control amplicon” or “IC amplicon” or “IC amplificationproduct” or variants thereof is meant an amplicon synthesized using aninternal control nucleic acid as the template in a nucleic acidamplification reaction.

As used herein, “apparatus” refers to the things necessary to carry outa purpose or for a particular use.

As used herein, a “holder” is a structural element for keeping somethingin place. For example, a holder may contain a tube, a multiwell plate, acapillary, or other reaction vessel. The holder may include mechanicalclips to retain the thing being held. Preferably, the holder of anapparatus useful for performing the invented assays will permit opticalaccess between a sample being held, such as a liquid-phase reactionmixture, and an optical detection mechanism.

As used herein, an “optical detection mechanism” refers to thecollection of components necessary for collecting optical signals from asample undergoing testing. Preferred examples of useful opticaldetection mechanisms include fluorometers and luminometers.

As used herein a “channel” of an energy sensor device, such as a deviceequipped with an optical energy sensor, refers to a defined band ofwavelengths that can be detected or quantified to the exclusion of otherbands of wavelengths. For example, one detection channel of aluminometer might be capable of detecting light energy emitted by one ormore chemiluminescent labels over a range of wavelengths as a singleevent. Light emitted during a chemiluminescent reaction can bequantified by a luminometer using relative light units (RLU), a unit ofmeasurement indicating the relative number of photons emitted by thesample at a given wavelength or band of wavelengths. Light emitted asthe result of fluorescence can be quantified as relative fluorescenceunits (RFU) at a given wavelength, or over a band of wavelengths.

As used herein, “single-channel detection” refers to a process wherebyone or more signals can be detected within a defined band of wavelengthsrepresented by one channel of an energy sensor device. If, for example,two detectable labels, each disposed on a different probe, both emitlight of characteristic wavelengths different from each other, and ifthose wavelengths are detected within a defined band of wavelengthscorresponding to one detection channel of an energy sensor device, thenthe detection would be described as “single channel detection.” Bysingle channel detection there is no distinction between which labelproduced the photon being detected when the photons arise from differentlabels, and have wavelengths falling within the detection range of thesingle detection channel.

As used herein, “single-read determination” refers to the process ofobtaining results from a detection step following a binding reactionbetween a probe and an analyte. By single-read determination it isunnecessary to change reaction conditions, such as probe hybridizationconditions, or to perform a secondary hybridization reaction.

As used herein, an “internal control signal” (sometimes referred to asan “IC signal”) is a measurable signal indicating the presence of aninternal control, or product thereof (e.g., such as an amplificationproduct) in a reaction mixture. The IC signal may be produced directlyby the internal control, for example if the internal control is labeled.Alternatively, the internal control signal may be produced by a probe(e.g., an internal control probe) that specifically interacts with theinternal control or product thereof. Preferred internal controls includeproteins and nucleic acids.

As used herein, an “analyte signal” is a measurable signal indicatingthe presence of an analyte, or product thereof (e.g., such as anamplification product) in a reaction mixture. The analyte signal may beproduced directly by the analyte, for example if the analyte is labeled.Alternatively, the analyte signal may be produced by a probe (e.g., ananalyte probe) that specifically interacts with the analyte. Preferredanalytes include proteins and nucleic acids.

As used herein, a “combined signal value” is a single value indicatingthe combination of detectable signals measured for an analyte and an ICin a single reaction mixture. A combined signal does not distinguish theinternal control signal from the analyte signal. For example, a combinedsignal value may be reported in RLU (relative light units) forchemiluminescent label(s), or in RFU (relative fluorescence units) forfluorescent label(s).

In certain multiplex assays that detect more than one analyte, thedifferent analytes may be detected by detecting or measuring,respectively, a “first analyte signal” and a “second analyte signal.”The presence or absence of the different analytes may be judged bycomparing the magnitudes of the respective signals with respectivecutoff values (e.g., “first” and “second” analyte cutoff values).

As used herein, a “threshold” or “threshold cutoff” or simply “cutoff”refers to a quantitative limit used for interpreting experimentalresults, where results above and below the cutoff lead to oppositeconclusions. For example, a measured signal falling below a cutoff mayindicate the absence of a particular target, but a measured signal thatexceeds the same cutoff may indicate the presence of that target. Byconvention, a result that meets a cutoff (i.e., has exactly the cutoffvalue) is given the same interpretation as a result that exceeds thecutoff.

As used herein, a “validity cutoff value” is a cutoff value used fordetermining whether or not a process step is valid (e.g., valid orinvalid). For example, an internal control signal that meets or exceedsa validity cutoff may indicate the process functioned as expected, andthat results of an assay incorporating that process are valid.Conversely, an internal control signal falling below the validity cutoffmay indicate the process did not function as expected, and that resultsof the assay are invalid.

As used herein, an “analyte cutoff value” is a cutoff value used forindicating the presence or absence of an analyte in a reaction mixtureor test sample. For example, an analyte signal that meets or exceeds ananalyte cutoff may indicate the presence of the analyte in a reactionmixture or test sample. Conversely, an analyte signal falling below theanalyte cutoff, if validated by process control, would indicate theabsence of the analyte.

As used herein, a “look-up table” refers to a collection of possiblecombinations of positive and negative results expressed relative to(e.g., < or ≥) a threshold cutoff value. Combinations in the collectionmay be associated with an interpretation that assigns positive ornegative status to the presence of an analyte in a sample undergoingtesting. Assay validity status also can be assigned. A look-up table canbe stored on computer-readable media, and conventionally is used fordecoding experimental results.

By “kit” is meant a packaged combination of materials, typicallyintended for use in conjunction with each other. Kits in accordance withthe invention may include instructions or other information in a“tangible” form (e.g., printed information, electronically recorded on acomputer-readable medium, or otherwise recorded on a machine-readablemedium such as a barcode for storing numerical values).

PREFERRED EMBODIMENTS

The analytical technique described herein, in certain respects, goesopposite earlier approaches used by many others. For example, unlike theabove-referenced U.S. Pat. No. 6,586,234, which describes IC validationusing a two-read approach that differentiates (a) analyte signal from(b) the combination of analyte signal and IC signal, the presentapproach never isolates these signals. More specifically, the presentapproach employs a single read method that does not require attributingthe origin or magnitude of signal arising from IC and analyte probes,even when the signals are detected using a single detection channel of adetection device, as may result from the use of identical labels on thetwo probes. Indeed, the present technique uses single-channel detectionfor simultaneously detecting, in a single reaction mixture, signalsproduced by both the IC probe and the analyte probe. The presenttechnique does not separate IC and analyte probes in separate detectionreactions, but instead combines the probes, and detects signals arisingtherefrom simultaneously. Again, the IC and analyte probesadvantageously can harbor either the same detectable label, or differentlabels that are detected using a single channel of a detection device.Where others may use a single cutoff for detecting signals from multipletargets detected using the same label, the present technique requiresthe use of separate cutoffs (e.g., so-called validity cutoff, andanalyte cutoff). Use of the plurality of separate cutoffs permitsavoidance of separate reads to distinguish signals arising from thedifferent probes. In accordance with the present technique, there are aplurality of threshold cutoffs, and there is a requirement that themagnitude of signal arising from detection of IC probe cannot exceed thecutoff used for indicating the presence of analyte. Stated differently,the maximum detectable IC signal cannot exceed the cutoff used forindicating the presence of analyte. In aggregate, these differencesdistinguish the present technique from earlier approaches.

Generally speaking, the techniques disclosed herein can be applied todetection of a variety of analytes, including: nucleic acids (e.g., DNAand RNA), proteins (e.g., antibodies, receptors for hormones or otherligands, etc.), as well as other molecules of biological interest.Particularly preferred analytes for identification by the disclosedmethods are nucleic acids that are detected using complementaryhybridization probes. The IC preferably is an exogenous, syntheticnucleic acid that is included in a reaction mixture being tested for thepresence of analyte nucleic acid prior to co-amplification with analytenucleic acid. Separate hybridization probes having different basesequences are used for detecting analyte and IC amplicons. The detectionstep is carried out at constant temperature. In a highly preferredembodiment, the different probes having specificity for the differenttarget nucleic acids (i.e., IC and analyte) are labeled with the samechemical species of detectable label. However, if different detectablelabels are used for labeling the different probes, signals produced bythe different labels must be detectable using a single channel of adetection device. Preferably, both detectable labels produce opticalsignals that are detected using a single channel of a detection device,where the channel is defined by a predetermined wavelength range.

Thresholds

One aspect of the present invention relates to the definition and use ofa plurality of threshold cutoffs for detectable signals that are usedfor identifying the presence or absence of an analyte in an assayvalidated by an IC. Preferably, when IC and analyte are detected usingdifferent probes that harbor the same chemical species of detectablelabel, there are two threshold cutoffs for a signal detected in a singlechannel of a detection instrument that detects analyte signal and ICsignal. The lower of the two threshold cutoffs (i.e., the “validitycutoff”) is used for validating the IC process control. The upper of thetwo threshold cutoffs is used for indicating the presence or absence ofanalyte in the sample undergoing testing. A signal that fails to meet orexceed the value of the validity cutoff indicates an invalid reactiondue to failure of the assay process. Such a situation may result frominhibition of an amplification step and/or detection step of the assay.A signal that exceeds the validity cutoff, but does not meet or exceedthe threshold cutoff for analyte detection indicates success of theamplification and detection steps of the assay, and further indicatesthe absence of analyte from the sample. This latter result can be scoredas “valid, analyte-negative” by the method disclosed herein. Finally,detection of a signal that exceeds both the validity cutoff and theanalyte threshold cutoff indicates the presence of analyte in the sample(i.e., an “analyte-positive” sample). When this is the case, there is noneed to report or question validity of the assay result. These featuresof the invention are illustrated in FIG. 1.

Success of the technique disclosed herein depends on certain generalrelationships between the allowable magnitudes of the signalsrepresenting detection of IC and analyte, and the plurality of thresholdcutoffs. Importantly, the validity cutoff must be distinct from, andlower than the threshold cutoff for analyte detection when the analyteand IC are detected using different probes harboring detectable labelsthat produce signals detectable in a single channel of a detectiondevice. In a highly preferred embodiment, the detectable labels are thesame detectable label (e.g., the same chemical species of fluorescentlabel, or chemiluminescent label, such as an AE label). Indeed, signalresulting from amplification and detection of IC that exceeds thevalidity cutoff should not ambiguously indicate the presence of analytedetected using probes harboring the same chemical species of detectablelabel. This may be ensured, for example, by requiring that the magnitudeof the signal resulting from detection of IC amplicons only (i.e., noanalyte being present in the reaction) has an upper limit threshold thatcannot be exceeded. In a particularly preferred approach, this isaccomplished by ensuring that the IC amplification and detectioncomponent of the disclosed assays are calibrated so that the IC signalcannot exceed the upper limit threshold cutoff in amplification reactionthat contains no analyte. This prevents false-positive resultsindicating the presence of analyte due to amplification and detection ofthe IC only. The threshold cutoff for detection of analyte is alwaysgreater than the validity cutoff. Detection of a signal that meets orexceeds the threshold cutoff for analyte detection automaticallyvalidates the analyte-positive assay result.

Many different approaches can be used to ensure that the maximum signalarising from IC detection (e.g., detection of IC amplicons) is below thethreshold cutoff for detection of analyte, when IC and analyte aredetected using a single chemical species of detectable label, ordifferent detectable labels that can be detected using a single channelof a detection device. For example, the specific activity (e.g.,measurable as units of detectable label per unit mass of probe) for ICamplicons can be reduced relative to the specific activity of probe usedfor detecting analyte amplicons. The amount or concentration ofIC-specific probe used in the hybridization reaction can be reducedrelative to the amount or concentration of analyte-specific probe. Thelabel disposed on the IC-specific probe can be selected to be lessefficiently detected relative to the label disposed on theanalyte-specific probe. In a highly preferred embodiment, the differentprobes used for detecting IC amplicons and analyte amplicons are labeledwith the same chemical species of detectable label (e.g., achemiluminescent label such as an acridinium ester label of a particularstructure, or alternatively a fluorescent label of a particularstructure), and the amount of IC-specific probe used for detecting ICamplicons is less than the amount of analyte-specific probe used fordetecting analyte amplicons. Of course, input amounts of IC templatenucleic acid used in co-amplification reactions also can be adjusted sothat the magnitude of the hybridization signal arising from detection ofIC amplicons is below the value of the analyte cutoff. For example, theinput amount of IC nucleic acid may be chosen to be no greater than tentimes the lower limit of detection for analyte, more preferably nogreater that three times the lower limit of detection for analyte, morepreferably no greater than two times the lower limit of detection foranalyte, and still more preferably no greater than the lower limit ofdetection for analyte. For example, the amount of IC template nucleicacid used in an amplification reaction preferably falls in the range offrom one-half to ten times the lower limit of detection for analyte inthe assay, still more preferably in the range of from one-tenth to onetimes the lower limit of detection for analyte in the assay.Combinations of any of these low input levels of IC template, togetherwith any of the above-described controlled amounts of probe also havebeen used successfully, and are within the scope of the presentdisclosure.

The foregoing discussion of threshold cutoffs is relevant to analysis ofIC and analyte when those targets are detected using a single chemicalspecies as the detectable label (e.g., same chemiluminescent label, samefluorescent label, etc.), or different chemical species of detectablelabels that can be detected using a single channel of a detectiondevice. Analysis of results obtained by this approach is illustrated inTable 1. Detection of a second analyte using a detectable labeldifferent from the one(s) used for detecting IC and the first analytecan be accomplished, and may involve the use of a different thresholdcutoff. Indeed, the threshold cutoff used for assessing the presence orabsence of the second analyte can be independent of the thresholdcutoffs used for assessing results for the first analyte and IC.Analysis of results obtained by this latter approach is illustrated inTable 2.

Certain Relationships Among Signal Magnitudes and Threshold Cutoffs

As stated elsewhere herein, there are a number of meaningfulrelationships among the magnitudes of signals representing detection ofinternal control and one or more analytes, and various respectivethreshold cutoffs. For example, in the context of an assay, andapparatus for performing the assay, that includes an internal controland first analyte, optionally including a second analyte, the magnitudeof a measured combined signal (e.g., produced by an internal controlprobe and first analyte probe) can be compared with a validity cutoffvalue and with a first analyte cutoff value. If the assay includesmeasurement of a second analyte signal (e.g., being produced by a secondanalyte probe, and being distinguishable from the combined signal), thenthat second analyte signal can be compared with a second analyte cutoffvalue. If the magnitude of the combined signal is greater than or equalto the validity cutoff value it is established by process control thatassay results are valid. If the magnitude of the combined signal isgreater than or equal to the first analyte cutoff value it isestablished that the first analyte is detected. If the magnitude of thecombined signal is less than the validity cutoff value it is notestablished by process control that assay results are valid. If thereaction mixture includes the second analyte probe, and if the magnitudeof the second analyte signal is greater than or equal to the secondanalyte cutoff value it is established by process control that assayresults are valid, and established that the second analyte is detected.If the reaction mixture includes the second analyte probe, and if themagnitude of the second analyte signal is less than the second analytecutoff value it is not established that the second analyte is detected,and it is not established by process control that assay results arevalid. Thus, when the second analyte signal is detected, that signalalso can serve to validate assay results, including negative results fordetection of the first analyte. Each of these determinations may beestablished by a processor or computer component of an apparatusaccording to the invention.

Examples of Threshold Establishment

Comparisons between threshold cutoffs (i.e., validity cutoff, andanalyte cutoff) and measured test signals representing combined signalsfor detection of IC plus analyte can be implemented by alternativeapproaches. In one preferred embodiment, predetermined threshold cutoffsare used for assessing test results and determining whether analyte ispresent or absent. In a different preferred embodiment, thresholdcutoffs are established using calibrators run on each different machinethat is to be used for testing.

Establishing threshold cutoffs specific for a particular machine and/orset of reagents may be carried out in different ways, but generally willemploy one or more calibrator standards (i.e., one or more standardscontaining known amounts of relevant nucleic acid to be amplified anddetected). Each calibration reaction includes the internal control. A“negative” calibrator can be used for establishing a validity cutoffthat must be exceeded by a signal representing detection of IC andanalyte for an assay to be regarded as valid. The negative calibratorreaction preferably includes IC template nucleic acid that can beamplified and detected, but does not include any analyte nucleic acid.One approach for establishing the value of the validity cutoff is tocalculate one-half (i.e., 50%) of the value of a signal measured in anegative calibrator run (i.e., amplification and detection procedure),or more preferably one-half of the value of an average of signalsmeasured in a plurality of negative calibrator amplification anddetection reactions. Any test reaction yielding a combined signal valuebelow this validity cutoff would be regarded as invalid (i.e.,indicating process failure) in the absence of validating resultsmeasured for a second analyte. Fractions of the negative calibratorresults other than one-half (e.g., 60%, 70%, etc.) may be alternativelybe chosen as the validity cutoff with good results. Any test reactionyielding a signal value above the validity cutoff would be regarded asvalid.

The upper threshold (i.e., the “analyte cutoff”) that must be exceededfor a result to be regarded as positive for analyte (i.e.,“analyte-positive”) in its simplest form also can be determined from theresult obtained using the negative calibrator reaction. The analytecutoff preferably will be at least one and one-half times the value ofthe signal measured for the negative calibrator trial (or the average ofnegative calibrator runs). More preferably, the analyte cutoff will beat least two times the value of the signal measured for the negativecalibrator trial. Still more preferably, the analyte cutoff isdetermined using results from negative calibrator trials, as well asfrom positive calibrator trials. For example, an analyte cutoff can bedetermined by increasing the value of a multiple of the negativecalibrator by a fractional amount (e.g., 10%, 20%, 30%, or in the rangeof from 10%-30%) of the value measured for a positive calibrator thatyields a signal representing detection of IC and analyte, where bothtargets are detected using single channel detection. This is illustratedin the Example, below.

Detection of Nucleic Acids

In a preferred embodiment, nucleic acid amplicons are detected insolution using solution-phase hybridization probes that are notimmobilized to a solid support when the hybridization signal isdetected. This is clearly different from arrayed detection formats, suchas nucleic acid microarrays, where interpretation of probe hybridizationresults depends on spatial separation of one probe from another. Aswell, the invented method can be practiced using an IC probe and ananalyte probe (e.g., each of these being a nucleic acid hybridizationprobe) that harbor identical chemical species of detectable label, orlabels that are similar enough to permit detection using a singledetection channel in a detection device. Notably, preferred proceduresdo not involve detection of a signal representing the presence ofanalyte only, without also detecting a signal representing the presenceof IC. Likewise, preferred procedures do not involve detection of asignal representing the presence of IC only, to the exclusion ofanalyte, when analyte also is available for detection. For example, incertain embodiments there is detected a cumulative signal indicating thepresence of both IC and analyte, meaning that IC signal and analytesignal are not detected separately. In this way, the present methoddiffers from certain other assay formats wherein analyte signal and ICsignal are detected separately.

Look-Up Tables

The method described herein conveniently can employ a look-up table forinterpreting results and determining the presence or absence of analytein a sample, as well as for validating assay integrity. Table 1represents a basic look-up table useful for interpreting results inaccordance with the disclosed method of detecting analyte and IC usingsingle channel detection of analyte and IC signals, as may be providedby different probes (i.e., separate probes for IC and analyte) labeledwith a single type of detectable label (i.e., identical labels on eachprobe). With reference to the arrangement of threshold cutoffs,detection of a signal that is below the threshold cutoff for analyte andalso below the validity cutoff indicates that the test is invalid.Conversely, detection of a signal that is below analyte cutoff, butabove the validity cutoff indicates that the test is valid, andanalyte-negative. Finally, a signal that is above the analyte cutoffindicates the test is analyte-positive. Thus, using as few as two probesharboring the same chemical species of detectable label can provideinsight into validity of an analytical process, as well as insight intothe presence or absence of an analyte.

TABLE 1 Analysis of Results Obtained Using a Single Label Species forDetecting IC and Analyte Signal Evaluation Magnitude of Signal Comparedto Cutoff Magnitude of Signal for Detection of Compared to ValidityAnalyte Cutoff Result for Analyte ≥Analyte cutoff >Validity cutoff,since Positive analyte cutoff is higher than validity cutoff <Analytecutoff ≥Validity cutoff Negative <Validity cutoff Invalid Assay

Multiplexing Advantages and Look-Up Tables

Another advantage of using signal magnitude (e.g., hybridization signalmagnitude) as a variable for distinguishing invalid reactions, validreactions indicating analyte-negative samples, and reactions indicatinganalyte-positive samples relates to the ability to detect multipleanalytes using only a small number of detectable labels. For example,when first analyte (Analyte-1) and IC nucleic acid templates areco-amplified and detected using different probes, each probe harboring alabel that can be detected using a first detection channel of adetection device (e.g., the first detectable labels being identical toeach other), an unrelated target (Analyte-2) can be detected using aprobe harboring a different detectable label, where that different labelcan be distinguished from the labels used on the Analyte-1 and IC probes(e.g., by kinetic resolution, or by detection using a second detectionchannel of the detection device, etc). When this is the case, a positiveresult for detection of Analyte-2 may also serve to validate assayresults. As well, Analyte-2 may be positively detected when the detectedsignal meets or exceeds a second analyte threshold, which may be thesame or different from the threshold cutoff that must be met or exceededto establish the presence of Analyte-1 in the sample. Thus, detection ofa signal that exceeds the threshold cutoff for detection of Analyte-2can validate the assay result (i.e., indicate that the assay componentsfunctioned as intended). In accordance with the method, even in theabsence of a detectable signal indicating hybridization of the Analyte-1and IC probes, an Analyte-2 signal that meets or exceeds the thresholdcutoff for detection of Analyte-2 can indicate the sample undergoingtesting included Analyte-2, but not Analyte-1, and that the assayresults are valid (i.e., valid, Analyte-1 negative; Analyte-2 positive).The logical analysis of results from a simple multiplex assay thatincludes Analyte-1 and IC probes labeled with a commonly detectablelabel(s) (i.e., the labels on the two probes being detectable using thesame detection channel in a detection device), together with anAnalyte-2 probe labeled with a second detectable label that isdistinguishable from the first label, is presented below in the form ofa look-up table (see Table 2).

TABLE 2 Analysis of Results Obtained Using Two Label Species forDetecting IC and Two Analytes Magnitude Signal 1 Evaluation of Signal 2Magnitude Compared of Signal1 to Cutoff Compared Result Result for toCutoff for Magnitude of for for Detection Detection of Signal 1 ComparedAnalyte- Analyte- of Analyte-2 Analyte-1 to Validity Cutoff 1 2≥Analyte-2 <Analyte-1 Valid whether signal 1 Negative Positive cutoffcutoff is ≥ or < since high signal 2 validates process control≥Analyte-1 Valid since Analyte-1 Positive Positive cutoff cutoff ishigher <Analyte-2 ≥Analyte-1 than validity cutoff Positive Negativecutoff cutoff <Analyte-1 ≥Validity cutoff Negative Negative cutoff<Validity cutoff Invalid

Useful Probe Labeling Systems and Detectable Moieties

Essentially any labeling and detection system that can be used formonitoring specific binding between a probe and an analyte can be usedin conjunction with the present invention. Included among the collectionof useful labels are radiolabels, enzymes, haptens, linkedoligonucleotides, chemiluminescent molecules, fluorescent moieties(either alone or in combination with “quencher” moieties), andredox-active moieties that are amenable to electronic detection methods.Preferred chemiluminescent molecules include acridinium esters of thetype disclosed by Arnold et al., in U.S. Pat. No. 5,283,174 for use inconnection with homogenous protection assays, and of the type disclosedby Woodhead et al., in U.S. Pat. No. 5,656,207 for use in connectionwith assays that quantify multiple targets in a single reaction. Thedisclosures contained in these patent documents are hereby incorporatedby reference. Preferred electronic labeling and detection approaches aredisclosed in U.S. Pat. Nos. 5,591,578 and 5,770,369, and the publishedinternational patent application WO 98/57158, the disclosures of whichare hereby incorporated by reference. Redox active moieties useful aslabels in the present invention include transition metals such as Cd,Mg, Cu, Co, Pd, Zn, Fe and Ru.

Particularly preferred detectable labels for probes in accordance withthe present invention are detectable in homogeneous assay systems (i.e.,where, in a mixture, bound labeled probe exhibits a detectable change,such as stability or differential degradation, compared to unboundlabeled probe). While other homogeneously detectable labels, such asfluorescent labels and electronically detectable labels, are intendedfor use in the practice of the present invention, a preferred label foruse in homogenous assays is a chemiluminescent compound (e.g., asdescribed by Woodhead et al., in U.S. Pat. No. 5,656,207; by Nelson etal., in U.S. Pat. No. 5,658,737; or by Arnold et al., in U.S. Pat. No.5,639,604). Particularly preferred chemiluminescent labels includeacridinium ester (“AE”) compounds, such as standard AE or derivativesthereof, such as naphthyl-AE, ortho-AE, 1- or 3-methyl-AE,2,7-dimethyl-AE, 4,5-dimethyl-AE, ortho-dibromo-AE, ortho-dimethyl-AE,meta-dimethyl-AE, ortho-methoxy-AE, ortho-methoxy(cinnamyl)-AE,ortho-methyl-AE, ortho-fluoro-AE, 1- or 3-methyl-ortho-fluoro-AE, 1- or3-methyl-meta-difluoro-AE, and 2-methyl-AE.

In some applications, probes exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,structures referred to as “Molecular Torches” are designed to includedistinct regions of self-complementarity (coined “the target bindingdomain” and “the target closing domain”) which are connected by ajoining region and which hybridize to one another under predeterminedhybridization assay conditions. When exposed to denaturing conditions,the two complementary regions (which may be fully or partiallycomplementary) of the Molecular Torch melt, leaving the target bindingdomain available for hybridization to a target sequence when thepredetermined hybridization assay conditions are restored. MolecularTorches are designed so that the target binding domain favorshybridization to the target sequence over the target closing domain. Thetarget binding domain and the target closing domain of a Molecular Torchinclude interacting labels (e.g., fluorescent/quencher) positioned sothat a different signal is produced when the Molecular Torch isself-hybridized as opposed to when the Molecular Torch is hybridized toa target nucleic acid, thereby permitting detection of probe:targetduplexes in a test sample in the presence of unhybridized probe having aviable label associated therewith. Molecular Torches are fully describedin U.S. Pat. No. 6,361,945, the disclosure of which is herebyincorporated by reference.

Another example of a self-complementary hybridization assay probe thatmay be used in conjunction with the invention is a structure commonlyreferred to as a “Molecular Beacon.” Molecular Beacons comprise nucleicacid molecules having a target complementary sequence, an affinity pair(or nucleic acid arms) holding the probe in a closed conformation in theabsence of a target nucleic acid sequence, and a label pair thatinteracts when the probe is in a closed conformation. Hybridization ofthe target nucleic acid and the target complementary sequence separatesthe members of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular Beaconsare fully described in U.S. Pat. No. 5,925,517, the disclosure of whichis hereby incorporated by reference.

Molecular beacons preferably are labeled with an interactive pair ofdetectable labels. Examples of detectable labels that are preferred asmembers of an interactive pair of labels interact with each other byFRET or non-FRET energy transfer mechanisms. Fluorescence resonanceenergy transfer (FRET) involves the radiationless transmission of energyquanta from the site of absorption to the site of its utilization in themolecule, or system of molecules, by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistances, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The “donor” is the moietythat initially absorbs the energy, and the “acceptor” is the moiety towhich the energy is subsequently transferred. In addition to FRET, thereare at least three other “non-FRET” energy transfer processes by whichexcitation energy can be transferred from a donor to an acceptormolecule.

When two labels are held sufficiently close that energy emitted by onelabel can be received or absorbed by the second label, whether by a FRETor non-FRET mechanism, the two labels are said to be in “energy transferrelationship” with each other. This is the case, for example, when amolecular beacon is maintained in the closed state by formation of astem duplex, and fluorescent emission from a fluorophore attached to onearm of the probe is quenched by a quencher moiety on the opposite arm.

Highly preferred label moieties for the invented molecular beaconsinclude a fluorophore and a second moiety having fluorescence quenchingproperties (i.e., a “quencher”). In this embodiment, the characteristicsignal is likely fluorescence of a particular wavelength, butalternatively could be a visible light signal. When fluorescence isinvolved, changes in emission are preferably due to FRET, or toradiative energy transfer or non-FRET modes. When a molecular beaconhaving a pair of interactive labels in the closed state is stimulated byan appropriate frequency of light, a fluorescent signal is generated ata first level, which may be very low. When this same probe is in theopen state and is stimulated by an appropriate frequency of light, thefluorophore and the quencher moieties are sufficiently separated fromeach other that energy transfer between them is substantially precluded.Under that condition, the quencher moiety is unable to quench thefluorescence from the fluorophore moiety. If the fluorophore isstimulated by light energy of an appropriate wavelength, a fluorescentsignal of a second level, higher than the first level, will begenerated. The difference between the two levels of fluorescence isdetectable and measurable. Using fluorophore and quencher moieties inthis manner, the molecular beacon is only “on” in the “open”conformation and indicates that the probe is bound to the target byemanating an easily detectable signal. The conformational state of theprobe alters the signal generated from the probe by regulating theinteraction between the label moieties. Examples of donor/acceptor labelpairs that may be used in connection with the invention, making noattempt to distinguish FRET from non-FRET pairs, includefluorescein/tetramethylrhodamine, IAEDANS/fluororescein, EDANS/DABCYL,coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPY FL,fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL, eosine/DABCYL,erythrosine/DABCYL, tetramethylrhodamine/DABCYL, Texas Red/DABCYL,CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2 and fluorescein/QSY7 dye. Thosehaving an ordinary level of skill in the art will understand that whendonor and acceptor dyes are different, energy transfer can be detectedby the appearance of sensitized fluorescence of the acceptor or byquenching of donor fluorescence. When the donor and acceptor species arethe same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Highly preferred quencher moieties that can be usedas another member of a donor-acceptor pair include DABCYL and the BLACKHOLE QUENCHER moieties which are available from Biosearch Technologies,Inc., (Novato, Calif.). Synthetic techniques and methods of bondinglabels to nucleic acids and detecting labels are well known in the art(e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989), Chapter 10; Nelson et al., U.S. Pat. No. 5,658,737; Woodhead etal., U.S. Pat. No. 5,656,207; Hogan et al., U.S. Pat. No. 5,547,842;Arnold et al., U.S. Pat. No. 5,283,174; Kourilsky et al., U.S. Pat. No.4,581,333), and Becker et al., European Patent App. No. 0 747 706.

Chemical Composition of Probes

Probes in accordance with the invention comprise agents able to complexwith analytes. Examples of useful probes include protein probes, such asantibody probes, and polynucleotide or nucleic acid probes.

Nucleosides or nucleoside analogs of preferred polynucleotide probescomprise nitrogenous heterocyclic bases, or base analogs, where thenucleosides are linked together, for example by phospohdiester bonds toform a polynucleotide. Accordingly, a probe may comprise conventionalribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), but also maycomprise chemical analogs of these molecules. The “backbone” of a probemay be made up of a variety of linkages known in the art, including oneor more sugar-phosphodiester linkages, peptide-nucleic acid bonds(sometimes referred to as “peptide nucleic acids” as described byHyldig-Nielsen et al., PCT Int'l Pub. No. WO 95/32305), phosphorothioatelinkages, methylphosphonate linkages or combinations thereof. Sugarmoieties of the probe may be either ribose or deoxyribose, or similarcompounds having known substitutions, such as, for example, 2′-O-methylribose and 2′ halide substitutions (e.g., 2′-F). The nitrogenous basesmay be conventional bases (A, G, C, T, U), known analogs thereof (e.g.,inosine or “I”; see The Biochemistry of the Nucleic Acids 5-36, Adams etal., ed., 11^(th) ed., 1992), known derivatives of purine or pyrimidinebases (e.g., N⁴-methyl deoxygaunosine, deaza- or aza-purines and deaza-or aza-pyrimidines, pyrimidine bases having substituent groups at the 5or 6 position, purine bases having an altered or a replacementsubstituent at the 2, 6 or 8 positions, 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Int'l Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). A probe may comprise onlyconventional sugars, bases and linkages found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more base analogs).

Preferred Nucleic Acid Amplification Reaction Formats

Preferred nucleic acid amplification methods may employ eitherthermocycling to alternately denature double-stranded nucleic acids andhybridize primers; or alternatively may employ isothermal reactionmechanisms. The polymerase chain reaction (Mullis et al., U.S. Pat. No.4,683,195; Mullis, U.S. Pat. No. 4,683,202; and Mullis et al., U.S. Pat.No. 4,800,159), commonly referred to as PCR, uses multiple cycles ofdenaturation, annealing of primer pairs to opposite strands, and primerextension to exponentially increase copy numbers of the target sequence.In a variation called RT-PCR, reverse transcriptase (RT) is used to makea complementary DNA (cDNA) from mRNA, and the cDNA is then amplified byPCR to produce multiple copies of DNA (Gelfand et al., “ReverseTranscription with Thermostable DNA Polymerases—High Temperature ReverseTranscription,” U.S. Pat. Nos. 5,322,770 and 5,310,652). Another methodis strand displacement amplification (Walker, G. et al. (1992), Proc.Natl. Acad. Sci. USA 89, 392-396; Walker et al., “Nucleic Acid TargetGeneration,” U.S. Pat. No. 5,270,184; Walker, “Strand DisplacementAmplification,” U.S. Pat. No. 5,455,166; and Walker et al. (1992)Nucleic Acids Research 20, 1691-1696), commonly referred to as SDA,which uses cycles of annealing pairs of primer sequences to oppositestrands of a target sequence, primer extension in the presence of a dNTPto produce a duplex hemiphosphorothioated primer extension product,endonuclease-mediated nicking of a hemimodified restriction endonucleaserecognition site, and polymerase-mediated primer extension from the 3′end of the nick to displace an existing strand and produce a strand forthe next round of primer annealing, nicking and strand displacement,resulting in geometric amplification of product. Thermophilic SDA (tSDA)uses thermophilic endonucleases and polymerases at higher temperaturesin essentially the same method (European Pat. No. 0 684 315). Otheramplification methods include: nucleic acid sequence based amplification(Malek et al., U.S. Pat. No. 5,130,238), commonly referred to as NASBA;one that uses an RNA replicase to amplify the probe molecule itself(Lizardi, P. et al. (1988) BioTechnol. 6, 1197-1202), commonly referredto as Qβ replicase; a transcription-based amplification method (Kwoh, D.et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173-1177); self-sustainedsequence replication (Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874-1878; Landgren (1993) Trends in Genetics 9, 199-202; andLee, H. et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)); and,transcription-mediated amplification (Kacian et al., “Nucleic AcidSequence Amplification Methods,” U.S. Pat. No. 5,480,784; and Kacian etal., U.S. Pat. No. 5,399,491), commonly referred to as TMA. For furtherdiscussion of known amplification methods see Persing, David H., 1993,“In Vitro Nucleic Acid Amplification Techniques” in Diagnostic MedicalMicrobiology: Principles and Applications (Persing et al., Eds.), pp.51-87 (American Society for Microbiology, Washington, D.C.). Otherillustrative amplification methods suitable for use in accordance withthe present invention include rolling circle amplification (RCA)(Lizardi, “Rolling Circle Replication Reporter Systems,” U.S. Pat. No.5,854,033); Helicase Dependent Amplification (HDA) (Kong et al.,“Helicase Dependent Amplification Nucleic Acids,” U.S. Pat. Appln. Pub.No. US 2004-0058378 A1); and Loop-Mediated Isothermal Amplification(LAMP) (Notomi et al., “Process for Synthesizing Nucleic Acid,” U.S.Pat. No. 6,410,278).

Preferred transcription-based amplification systems of the presentinvention include TMA, which employs an RNA polymerase to producemultiple RNA transcripts of a target region (e.g., Kacian et al., U.S.Pat. Nos. 5,480,784 and 5,399,491; and Becker et al., “Single-PrimerNucleic Acid Amplification Methods,” U.S. Pat. Appln. Pub. No. US2006-0046265 A1). Transcription mediated amplification (TMA) uses a“promoter oligonucleotide” or “promoter-primer” that hybridizes to atarget nucleic acid in the presence of a reverse transcriptase and anRNA polymerase to form a double-stranded promoter from which the RNApolymerase produces RNA transcripts. These transcripts can becometemplates for further rounds of TMA in the presence of a second primercapable of hybridizing to the RNA transcripts. Unlike PCR, LCR or othermethods that require heat denaturation, TMA is an isothermal method thatuses an RNAse H activity to digest the RNA strand of an RNA:DNA hybrid,thereby making the DNA strand available for hybridization with a primeror promoter-primer.

In one illustrative TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence whichbecomes functional when double-stranded, located 5′ of a target-bindingsequence, which is capable of hybridizing to a binding site of a targetRNA at a location 3′ to the sequence to be amplified. A promoter-primermay be referred to as a “T7-primer” when it is specific for T7 RNApolymerase recognition. Under certain circumstances, the 3′ end of apromoter-primer, or a subpopulation of such promoter-primers, may bemodified to block or reduce primer extension. From an unmodifiedpromoter-primer, reverse transcriptase creates a cDNA copy of the targetRNA, while RNAse H activity degrades the target RNA. A secondamplification primer then binds to the cDNA. This primer may be referredto as a “non-T7 primer” to distinguish it from a “T7-primer.” From thissecond amplification primer, reverse transcriptase creates another DNAstrand, resulting in a double-stranded DNA with a functional promoter atone end. When double-stranded, the promoter sequence is capable ofbinding an RNA polymerase to begin transcription of the target sequenceto which the promoter-primer is hybridized. An RNA polymerase uses thispromoter sequence to produce multiple RNA transcripts (i.e., amplicons),generally about 100 to 1,000 copies. Each newly-synthesized amplicon cananneal with the second amplification primer. Reverse transcriptase canthen create a DNA copy, while the RNAse H activity degrades the RNA ofthis RNA:DNA duplex. The promoter-primer can then bind to the newlysynthesized DNA, allowing the reverse transcriptase to create adouble-stranded DNA, from which the RNA polymerase produces multipleamplicons.

Preferred Analyte Polynucleotides

The present invention is not limited to the use of particular nucleotidesequences, nucleic acid analytes, primers or hybridization probes. Thus,the specific oligonucleotides used in the Examples are not essentialfeatures of the present invention.

Preferred analyte polynucleotides include nucleic acids fromdisease-causing organisms, including viruses, bacteria, fungi andprotozoa. Examples of highly preferred analyte polynucleotides fromviruses are nucleic acids from the human immunodeficiency viruses (HIV-1and HIV-2), the hepatitis B virus (HBV), the hepatitis C virus (HCV),human papillomaviruses (HPV), Dengue virus (DEN), Chikungunya virus(CHIKV), etc. Preferred analyte polynucleotides from bacteria, fungi andprotozoa that can be quantitated according to the methods disclosedherein include the ribosomal RNAs (rRNA). Examples of bacteria that arehighly preferred as sources of analyte polynucleotides include Chlamydiatrachomatis (Gram-negative cells that are obligate intracellularorganisms), members of the genus Campylobacter (C. jejuni, C. coli, C.laridis), members of the genus Enterococcus (E. avium, E. casseliflavus,E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, E. mundtii,E. pseudoavium, E. malodoratus, and E. raffinosus), Haemophilusinfluenzae, Listeria momocyto genes, Neisseria gonorrhoeae,Staphylococcus aureus, Group B Streptococci, Streptococcus pneumoniae,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium gordonae, Mycobacterium kansasii. Examplesof fungi that are highly preferred as sources of analyte polynucleotidesinclude: Blastomyces dermatitidis, members of the genus Candida (C.albicans, C. glabrata, C. parapsilosis, C. diversus, C. tropicalis, C.guilliermondii, C. dubliniensis), Histoplasma capsulatum, Coccidioidesimmitis. Examples of protozoa that are highly preferred as sources ofanalyte polynucleotides include blood and tissue protozoa, such asmembers of the genus Plasmodium (P. malariae, P. falciparum, P. vivax),as well as protozoa which infect the gastrointestinal tract such asGiardia lamblia and Cryptosporidium parvum.

The disclosed method also can be used for detecting nucleic acids thatare of human origin, such as mRNAs that are over-expressed orunder-expressed in disease states, including cancers. One example of agene that is present at an increased copy number in breast and ovarianadenocarcinomas is the HER-2/neu oncogene which encodes a tyrosinekinase having certain features in common with the epidermal growthfactor receptor (EGFR). U.S. Pat. No. 4,968,603 describes the value ofmeasuring the increased copy number of the HER-2/neu gene, or theHER-2/neu mRNA as a tool for determining neoplastic disease status.Thus, for example, the method described herein can be employed inquantitative nucleic acid amplification protocols whereby the cellularcontent of HER-2/neu polynucleotides is determined.

Indeed, the method described herein is broadly applicable to numerousnucleic acid targets and is easily extended to procedures forquantifying any given analyte polynucleotide in a test sample.

Apparatus and Transformation Alternatives

An apparatus useful for carrying out the disclosed method typically willinclude a holder for the sample undergoing testing, an optical detectionmechanism that detects and/or quantifies signals indicating themagnitude of probe binding for IC and analyte probes, and a processor(e.g., a computer) that analyzes data and determines whether the analyteis present or absent, or even whether such a determination is possible.A preferred example of such an apparatus is a nucleic acid amplificationand detection device. The method implemented on the apparatus mayinvolve hybridization probes, and the detection of optical signalsgenerated by detectable labels may take place at constant temperature(e.g., ambient temperature, or a different constant temperature). Inaccordance with the invention, it is not a requirement to gather opticalsignal data at different temperatures in order to determine the presenceor absence of analyte in a test sample. A preferred structure thatmaintains the constant temperature is a temperature-controlledincubator. The temperature-controlled incubator is optional if signaldetection takes place at ambient temperature. Of course, the apparatusmay also include a temperature-controlled incubator for amplifyingnucleic acids, although that incubator need not be used during the stepof detecting optical signals used for determining the presence orabsence of analyte in a sample. For example, the holder may be containedwithin the temperature-controlled incubator to maintain its constanttemperature, or may be independent of the temperature-controlledincubator which serves in steps related to nucleic acid amplification orsome other process. Preferably, the apparatus is configured to hold amultiwell plate or a plurality of tubes. In one embodiment, the opticaldetection mechanism includes a luminometer that detects light outputfrom chemiluminescent reactions. In a different embodiment, the opticaldetection mechanism includes a fluorescence detector (i.e, afluorometer). In the case of nucleic acid analysis, detection ofhybridization signals preferably takes place at the conclusion of anamplification reaction, which is sometimes referred to as “end-pointdetection.” This is distinguished from real-time detection, wherein thedetection step is performed continuously or periodically as theamplification reaction is taking place. Preferably, the addition ofprobes specific for IC and analyte nucleic acids (e.g., amplificationproducts) to a nucleic acid amplification reaction mixture is performedby an automated testing instrument. In a generally preferred embodiment,the apparatus that carries out the disclosed method typically willinclude a computer or processor programmed with software instructions,or be capable of executing software instructions, for determiningwhether a test reaction is invalid, whether a test result is valid andnegative for the presence of analyte (i.e., the sample undergoingtesting does not include analyte), or whether a sample undergoingtesting is positive for the presence of analyte.

In certain instances, transformation of reagents (e.g.,deoxyribonucleotide triphosphates, and/or ribonucleotide triphosphates)into amplicons also is preferred when practicing the present invention.This may involve contacting a template nucleic acid with one or morepriming oligonucleotides (e.g., “primers”), and then enzymaticallyextending the priming oligonucleotides in a template-dependent fashion.

In certain other instances, transformation of reagents may involvetransformation of an indicator reagent to a detectable form, where thatdetectable form indicates the presence of IC or analyte in a startingsample or reaction mixture.

Software

Software products, whether in the form of machine-readable instructionsrecorded in tangible form (e.g., a machine-readable medium such as adisk having instructions recorded thereon using electronic, magnetic oroptical data storage), or loaded into a device that is a component of anapparatus for processing samples and acquiring results (e.g., a nucleicacid amplification device that performs probe hybridization anddetection), represent part of the subject matter embraced by the presentdescription. As well, a device for processing samples and acquiringresults (e.g., a nucleic acid amplification device that performs probehybridization and detection) that operates using the software also isembraced by the present description. Of course, the software can beloaded into a general purpose computer linked to the device forprocessing samples and acquiring results. Alternatively, the software beloaded into a computing device that is an integral component of thedevice for processing samples and acquiring results.

Calibration Software

The software feature of the invention optionally may includeinstructions for processing input results from one or more calibrationstandards. As a result there will be established a validity cutoff andan analyte cutoff, where these cutoffs are useful for determiningwhether an assay result is valid or invalid, and whether a sampleundergoing testing included, or did not include an analyte. Moreparticularly, the software processes results from a negative calibrator(i.e., a calibration standard that does not include any added analyte).Highly preferred software applications regard detection of nucleicacids, using techniques that involve nucleic acid amplificationprocedures. Of course, a nucleic acid amplification reaction carried outusing the negative calibrator will include the internal control, whetheras a component of the calibrator or added separately.

Preferred software is capable of establishing threshold cutoffs fordetermining validity of the assay process, as well as determining thepresence or absence of analyte in a sample undergoing testing. Theinstrument used for performing procedures preferably includes atemperature-controlled incubator in which nucleic acid amplificationreactions take place. More preferably, the instrument is furtherconfigured for performing nucleic acid hybridization reactions (e.g., atthe conclusion of amplification reactions), and detecting probe hybrids.The software is generally capable of receiving quantitative inputs fromone or more negative calibrator trials, where each trial includes areaction (e.g., nucleic acid amplification, and probe hybridization anddetection) carried out using IC in the absence of added analyte, andwhere reaction products of the negative calibrator trials are detectedby single channel detection. In this detection format, signalsindicating the presence of IC-specific and analyte-specific reactionproducts are quantified without distinguishing signals specific foreither of the two reaction products. The software is further capable ofestablishing a validity cutoff having a value less than 100% of themagnitude of the combined IC signal plus analyte signal measured for thenegative calibrator trial(s). Test reactions (i.e., reactions carriedout using test samples) yielding a combined IC plus analyte signal of amagnitude that meets or exceeds the validity cutoff will be judged asvalid (i.e., demonstrating that all assay process steps werefunctional). Test reactions yielding a combined IC plus analyte signalof a magnitude less than the validity cutoff will be judged as invalid.The software is further capable of receiving results from one or morepositive calibrator trials, where each trial includes a reaction (e.g.,nucleic acid amplification, and probe hybridization and detection)carried out using IC and a predetermined amount of analyte that yieldsdetectable reaction products for both IC and analyte, and where productsof the positive calibrator trials are, like the products of the negativecalibrator trial(s), detected by single channel detection. The softwareis further capable of establishing an analyte cutoff having a valuegreater than 100% of the magnitude of the combined IC plus analytesignal measured for the negative calibrator trial(s), and optionallyalso a fractional amount less than 100% of the positive calibratortrial. Test samples yielding a combined IC plus analyte signal having amagnitude that meets or exceeds the analyte cutoff will indicate thatthe sample undergoing testing is includes the analyte. Test samplesyielding a combined IC plus analyte signal of a magnitude less than theanalyte cutoff will indicate that the sample undergoing testing does notinclude analyte.

A noteworthy feature of the calibration software component of thepresent disclosure is the fact that two cutoffs are established, andthat these cutoffs are established using quantitative signal data basedon a single reading of a combined signal, where the combined signalincludes contributions from IC and analyte, but where the combinedsignal makes no distinction between the origin of the signalcontributions. Instead, the software is capable of determining thatanalyte is present in a test sample by comparing the combined signalagainst the determined threshold cutoffs.

Analytical Software

Software for analyzing experimental data obtained in connection with theinvented technique is able to determine whether the magnitude of acombined IC and analyte signal is above or below a plurality ofthreshold cutoffs. For example, preferred software instructions specifyperformance of a step to determine whether the magnitude of the combinedsignal meets or exceeds a threshold (i.e., the analyte cutoff) fordetermining the presence of analyte in a reaction mixture. If thecombined signal meets or exceeds the analyte cutoff, then the softwaremay output a result indicating that the sample undergoing testingincludes the analyte. Conversely, if the magnitude of the combinedsignal does not meet or exceed the analyte cutoff (i.e., falls below theanalyte cutoff), then the software also can instruct comparison of themagnitude of the combined signal with a validity cutoff to determinewhether assay results are valid or invalid. Here, a result wherein themagnitude of the combined signal is below the validity cutoff will beinterpreted as indicating an invalid assay. This may require that thetest is repeated, and the software may indicate the test is invalid. Onthe other hand, if the magnitude of the combined signal meets or exceedsthe validity cutoff, then the software interprets the assay result asbeing valid. Again, if the magnitude of the combined signal meets orexceeds the validity cutoff, but does not meet or exceed the analytecutoff, then the software instructs an output result indicating that thesample undergoing testing does not include the analyte. This result willbe considered valid, meaning that the conclusion regarding absence ofanalyte is accurate, and not due to failure of some assay component. Thelogic of this processing tree is embodied in the look-up table appearingin Table 1.

In addition to the above, preferred software instructions further canaccommodate interpretation of results obtained for an IC-validated assaythat detects a plurality of analytes. To simplify description of thisaspect of the software, the first analyte (i.e., “Analyte-1”) and IC, oramplicons arising therefrom, are detected using a “combined firstsignal” that is a combined IC plus Analyte-1 signal indicating detectionof these targets. Similarly, a “second signal” is used for detecting thesecond analyte (i.e., “Analyte-2”), or amplicons arising therefrom.Optionally, a third analyte (i.e, “Analyte-3”) also can be detected by a“combined second signal,” where this signal indicates the presence ofeither Analyte-2 or Analyte-3, without distinguishing one from theother. The software can receive input information for the second signal,and compare the magnitude of the second signal with a threshold cutofffor detection of Analyte-2 (i.e., the “Analyte-2 cutoff”). The Analyte-2cutoff optionally can be different from a threshold cutoff used fordetection of Analyte-1 (i.e., the “Analyte-1 cutoff”), since Analyte-2typically will be detected using a label that is distinguished from thelabel used for detecting Analyte-1. Here the software can instructperformance of a step to determine whether the magnitude of the secondsignal is above or below the Analyte-2 cutoff. If the magnitude of thesecond signal meets or exceeds the Analyte-2 cutoff, then the softwarereports that Analyte-2 is present in the sample undergoing testing. Ifthe magnitude of the second signal is below the Analyte-2 cutoff, theresult alternatively could mean that the sample undergoing testing didnot include Analyte-2, or that the result is invalid due to inhibitionof an assay process step. Regardless of whether the second signal isabove or below the Analyte-2 cutoff, the software preferablyinterrogates the magnitude of the combined first signal. Here again,preferred software instructions specify performance of a step todetermine whether the magnitude of the combined first signal meets orexceeds the Analyte-1 cutoff. If the combined first signal meets orexceeds the Analyte-1 cutoff, then the software may output a resultindicating that the sample undergoing testing includes Analyte-1. Sincethe Analyte-1 cutoff is higher than the validity cutoff, the Analyte-1positive result will automatically validate the assay results, meaningthat a second signal falling below the Analyte-2 cutoff will beinterpreted by the software as validating the Analyte-2 negative result.In this case the software can generate an output indicating that thesample undergoing testing includes Analyte-1, but does not includeAnalyte-2. If the magnitude of the combined first signal does not meetor exceed the Analyte-1 cutoff (i.e., falls below the Analyte-1 cutoff),then the software also can instruct comparison of the magnitude of thecombined first signal with a validity cutoff to determine whether assayresults are valid or invalid. Here, a result wherein the magnitude ofthe combined first signal is below the validity cutoff will beinterpreted by the software as indicating the assay results are invalidonly if the second signal also falls below the Analyte-2 cutoff. If themagnitude of the combined first signal is below the validity cutoff, andif the magnitude of the second signal meets or exceeds the Analyte-2cutoff, then the software reports that the sample undergoing testingincludes Analyte-2, but does not include Analyte-1. In this case thesecond signal can serve to validate assay results, even when the firstsignal is below the validity cutoff. Likewise, if the combined firstsignal meets or exceeds the validity cutoff, but falls below theAnalyte-1 cutoff, that result will validate the assay results, meaningthat the sample undergoing testing does not include Analyte-1. Whetherthe sample includes Analyte-2 will depend on whether the magnitude ofsecond signal meets or exceeds the Analyte-2 cutoff (in which case thesample includes Analyte-2), or whether the magnitude of the secondsignal falls below the Analyte-2 cutoff (in which case the sample doesnot include Analyte-2). The logic of this processing tree is embodied inthe look-up table appearing in Table 2.

Illustrative Examples

Following there is an exemplary case where an IC polynucleotide andoptionally also distinct first and/or second analyte polynucleotides(i.e., termed, “Analyte-1” and “Analyte-2”) were amplified and detected.More specifically, at the conclusion of the amplification reaction theIC amplicon, Analyte-1 amplicon, and Analyte-2 amplicon, if present,were all detected using target-specific hybridization probes. Probesspecific for IC and Analyte-1 were labeled with the same chemicalspecies of chemiluminescent label (i.e., an AE label). The probespecific for Analyte-2 amplicons was labeled with a second AE label thatwas distinguishable from the label used for detecting IC amplicons andAnalyte-1 amplicons. The magnitude of the combined probe hybridizationsignal for IC and Analyte-1 amplicon was measured and compared againsttwo threshold cutoffs to determine assay validity, and the presence orabsence of Analyte-1 nucleic acids in the reaction. In accordance withthe invention, a combined signal meeting or exceeding the lower of thesethresholds (i.e., the validity cutoff) indicates that amplification anddetection took place, thereby validating the assay (i.e., “assayvalid”). Conversely, a combined signal lower than the validity cutoffindicates the procedure failed, and the assay result is declared“invalid.” A combined signal meeting or exceeding a second threshold(i.e., the Analyte-1 cutoff), where the second threshold is higher thanthe first threshold, indicates that Analyte-1 was present in the sampleundergoing testing (i.e., Analyte-1 positive). A combined signal thatexceeds the validity cutoff but not the Analyte-1 cutoff indicates thesample is negative for Analyte-1, and that the assay result is valid(i.e., assay valid; Analyte-1 negative). Analyte-2 was detectedindependently by comparing the hybridization signal for Analyte-2specific probe against a threshold cutoff specific for that target(i.e., the Analyte-2 cutoff). A signal arising from the Analyte-2specific probe meeting or exceeding the Analyte-2 cutoff indicates thatAnalyte-2 was present in the sample undergoing testing. Notably, anysample that was positive for Analyte-2 was considered valid, regardlessof the signal detected for IC plus Analyte-1 amplicons. Finally,detection of a combined hybridization signal for IC and Analyte-1meeting or exceeding the Analyte-1 cutoff, together with a signalarising from the Analyte-2 specific probe meeting or exceeding theAnalyte-2 cutoff indicates that the sample undergoing testing includedboth Analyte-1 and Analyte-2.

Methods employing the acridinium ester labels described below are knownin the art of nucleic acid labeling. Indeed, Nelson et al., in U.S. Pat.No. 5,658,737 described simultaneously detecting multiple specificnucleic acid sequences using hybridization probes harboring kineticallydistinguishable chemiluminescent labels. Nelson et al., specificallyemployed different labels to distinguish hybridization of differenttarget-specific probes. Linnen et al., in published U.S. Pat. Appl. No.2004/0029111 described the use of hybridization probe cocktails fordetecting amplified viral nucleic acid targets, and in some casesinternal control amplicons. Here the kinetically distinguishable labelswere used for discriminating internal control amplicons from analyteamplicons. Trials wherein collections of probes were used for detectingviral amplicons were judged as positive or negative using a singlethreshold cutoff. The IC-validated assays disclosed by Linnen et al.,always required one label for detecting IC amplicons, and a differentlabel for detecting analyte amplicons. As will be apparent from thefollowing description, the disclosed technique includes a newarrangement of detectable labels on probes (e.g., nucleic acid probes)in a single reaction mixture, where that arrangement would havedelivered ambiguous results using methods previously disclosed byothers.

Example 1 describes an IC-validated assay capable of detecting a firstanalyte nucleic acid using a single read channel, and only a singlespecies of detectable label. The described assay is further capable ofdetecting a second analyte using a second species of detectable label,where the second label species is distinguishable from the first labelspecies. In this instance, the IC (i.e., a sequence essentiallyidentical to Analyte-1 except for a scrambled internal probe-bindingsequence) and Analyte-1 templates both amplified using a shared pair ofprimers, thereby defining a competitive IC amplification (i.e.,Analyte-1 and IC being amplified by shared primers). However, anon-competitive IC system (i.e., Analyte-1 and IC nucleic acids beingamplified by unrelated primers) can be substituted, and falls within thescope of the present method and apparatus. Notably, the assay used inthis illustration exhibited 95% positive detection of Analyte-1 andAnalyte-2 when the respective targets were present at 30copies/reaction.

Example 1

IC-Validated Assay for Detecting Two Different Analyte PolynucleotidesUsing Single Channel Read of Analyte and Process Control Signals

In vitro synthesized transcripts served as templates for amplificationin conventional TMA reactions. Negative control samples were representedby 400 μl volumes of specimen transport medium (STM) containing no addedAnalyte-1 or Analyte-2 nucleic acid. Test samples were represented by400 μl volumes of STM containing either 100 copies of an in vitrotranscript for Analyte-1, 100 copies of an in vitro transcript forAnalyte-2, or the combination of 10⁷ copies of the Analyte-1 in vitrotranscript and 10⁴ copies of the Analyte-2 in vitro transcript. STM is aphosphate-buffered detergent solution which, in addition to lysingcells, protects released RNAs by inhibiting the activity of RNases thatmay be present in the test sample. Aliquots (100 μl) of target-capturereagent (TCR) containing 200 copies of in vitro synthesized ICtranscripts were added to each sample, and mixed gently. This ensuredthat each reaction received the IC template nucleic acid. The TCRincluded magnetic particles (Seradyn, Inc.; Indianapolis, Ind.)displaying surface oligo(dT)₁₄; and a target-capture oligonucleotidehaving a stretch of poly(dA) joined to a sequence complementary toeither the IC and Analyte-1 nucleic acids, or to the Analyte-2 nucleicacid. Capture reaction mixtures were incubated sequentially at 62° C.for 30 minutes, and room temperature for 30 minutes to allow formationof hybridization complexes made up of target:captureoligomer:immobilized probe on the solid support particles. Hybridizationcomplexes on the particles were separated from other sample componentsby applying magnetic force to the outside of the vessel, aspirating awayother sample components that were not immobilized to the particles, andwashing the hybridization complexes on the particles using standardlaboratory procedures. Notably, the IC and Analyte-1 transcripts hadidentical sequences except for a scrambled region between primer bindingsites. These native and scrambled sequences served as probe bindingsites during the hybridization and detection procedure carried out atthe conclusion of the amplification step. Analyte-2 transcripts hadsequences that amplified using an independent primer set, whereamplification products were not detected by probes used for detectingeither IC or Analyte-1.

Amplification reactions were prepared by combining the purified magneticbead complexes from individual tubes with 75 μl aliquots of anamplification reagent and 200 μl of an inert oil overlay to controlevaporation. The TMA reactions were carried out essentially as describedby Kacian et al., in U.S. Pat. No. 5,399,491. The disclosure of thisU.S. patent is incorporated by reference. The amplification reagentincluded a pH-buffered mixture of salts, cofactors, deoxyribonucleotidetriphosphates (i.e., four dNTPs), and ribonucleotide triphosphates(i.e., four NTPs). The amplification reagent further included a T7promoter-primer and a non-T7 primer, where the combination was capableof amplifying both IC and Analyte-1 nucleic acid templates. Alsoincluded in the amplification reagent were a T7 promoter-primer andnon-T7 primer, where the combination was capable of amplifying Analyte-2nucleic acid. Contents of the tubes were mixed gently, heated briefly to62° C., and then equilibrated to 42° C. Next, reaction mixtures werecombined with 25 μl aliquots of an enzyme reagent, and then incubated at42° C. for an additional 60 minutes to permit amplification. The enzymereagent included Moloney murine leukemia virus (“MMLV”) reversetranscriptase and T7 RNA polymerase. Reactions resulted in production ofamplified DNA and RNA strands when appropriate template polynucleotideswere present.

At the conclusion of the 60 minute incubation period, amplificationreaction mixtures were subjected to probe hybridization assays.Oligonucleotide probes were prepared using 2′-methoxy (2′-OMe)nucleotide analogs, and labeled with acridinium ester according toprocedures that will be familiar to those having an ordinary level ofskill in the art. In this instance, the probe specific for IC ampliconsand the probe specific for Analyte-1 amplicons were both labeled withortho-fluoro AE, which is sometimes referred to as a “flasher” becauseof its rapid kinetic properties during chemiluminescent emission oflight. The probe specific for Analyte-2 amplicons was labeled with2-methyl AE, which is sometimes referred to as a “glower” because of itspersistent light production kinetic properties relative to the flasher.The detectable labels were joined to oligonucleotide structures byinternally disposed non-nucleotide linkers according to proceduresdescribed in U.S. Pat. Nos. 5,585,481 and 5,639,604, the disclosures ofthese patents are incorporated by reference. Hybridization reactionswere carried out by combining the 100 μl amplification reaction volumeswith 100 μl of a buffered probe reagent that included the threeoligonucleotide probes dissolved in a succinate-buffered detergentsolution. More specifically, the probe reagent was added to each tube,vortexed, and incubated in a water bath at 62° C. for 15 minutes.Following completion of the probe hybridization step, 250 μl ofselection reagent (a borate-buffered solution containing a surfactant)was added to each reaction tube. Tubes were vortexed, and then incubatedat 62° C. for 10 minutes. After removal from the 62° C. incubator, thetubes were cooled to 19-27° C. for 10-75 minutes and then placed in aLEADER HC+ luminometer (Gen-Probe Incorporated; San Diego, Calif.)configured for automatic injection of 0.1% hydrogen peroxide and 1 mMnitric acid; followed by injection of a solution containing 1 N NaOH.The combined IC flasher signal and Analyte-1 flasher signal in eachreaction was discriminated from the Analyte-2 glower signal by thedifferential kinetics of light emission, essentially as described byNelson et al., in U.S. Pat. No. 5,658,737. The disclosure of this U.S.patent is incorporated by reference. Software receiving inputs from theluminometer differentiated between the flasher and glower signals, andreported results for the chemiluminescent reactions in relative lightunits (RLU). Again, this procedure permitted assignment of signalcontributions due to: (1) the combination of IC plus Analyte-1hybridization; and (2) Analyte-2 hybridization. There was no distinctionbetween the signal arising from the IC probe and the Analyte-1 probe.

Although only two calibrators (i.e., the below described first andsecond calibrators) would generally be used for establishing cutoffs inassays intended for detecting a single analyte (e.g., Analyte-1) with ICvalidation, three calibrators were used for illustrating the techniquethat additionally permitted detection of the second analyte (Analyte-2).More specifically, calibration reactions were carried out to establishthe validity cutoff, the Analyte-1 cutoff, and the Analyte-2 cutoff. Thefirst calibration standard (i.e., “Cal(1)”) was a negative calibratorconsisting of STM buffer, and did not include added Analyte-1 orAnalyte-2 nucleic acid. The second calibration standard (i.e., “Cal(2)”)was a positive calibrator that included Analyte-1 transcripts in STMbuffer, in an amount that provided 400 copies/reaction. The secondcalibrator did not include any added Analyte-2 transcripts. The thirdcalibration standard (i.e., “Cal(3)”) was a positive calibrator thatincluded Analyte-2 transcripts in STM buffer, in an amount that provided300 copies/reaction. The third calibrator did not include any addedAnalyte-1 transcripts. It was established ahead of time using standardlaboratory procedures that will be familiar to those having an ordinarylevel of skill in the art that these input amounts of Analyte-1 andAnalyte-2 templates resulted in substantially saturating levels ofhybridization signal in the probe hybridization and detectionprocedures. Trials including the calibration standards were processedusing the target capture, amplification, and detection proceduresdescribed above. Calibration reactions were performed in replicates ofthree.

Table 3 summarizes results from calibration reactions used forestablishing the validity cutoff, the Analyte-1 cutoff, and theAnalyte-2 cutoff. The average flasher signal value determined using thenegative calibrator (i.e., Cal(1)) was multiplied by 0.5 to establish avalidity cutoff. The Analyte-1 cutoff was established by doubling theaverage flasher signal value determined using the negative calibrator,and then adding 10% of the average flasher signal value determined usingthe second calibrator (i.e., Cal(2)). The Analyte-2 cutoff wasestablished by arbitrarily calculating 18% of the average glower signalvalue determined using the third calibrator (i.e., Cal(3)), and thenadding the value of the average glower signal measured for Cal(1).

TABLE 3 Establishing Threshold Cutoffs Calibrator Avg. Flasher Avg.Glower Determined Cutoff ID Signal (RLU) Signal (RLU) (RLU value) Cal(1)207,910 0 Validity Cutoff (103,955 flasher RLU) Cal(2) 2,341,958 0Analyte-1 Cutoff (650,016 flasher RLU) Cal(3) 0 1,962,929 Analyte-2Cutoff (353,327 glower RLU)

Table 4 and FIG. 2 summarize results obtained using the test samples inreplicates of ten, and interpreted in view of the threshold cutoffspresented in Table 3. The negative control trials yielded a combinedaverage hybridization signal for the IC plus Analyte-1 flasher probesthat exceeded the validity cutoff, thereby establishing that the assayresult was valid. However, the magnitude of this signal was below theAnalyte-1 cutoff, and so indicated the test sample was negative for thepresence of Analyte-1, as expected. Likewise, the Analyte-2 glowersignal was below the Analyte-2 cutoff, thereby indicating the testsample was negative for Analyte-2, also as expected. Trials conductedusing 100 copies/reaction of the Analyte-1 transcript, and no Analyte-2transcript, yielded a combined average hybridization signal for the ICplus Analyte-1 flasher probes that exceeded both the validity cutoff andthe Analyte-1 cutoff, thereby establishing that the assay result wasvalid and that the test sample was positive for Analyte-1. These sametrials yielded average glower signals below the Analyte-2 cutoff,thereby indicating the test sample was negative for Analyte-2. Trialsconducted using 100 copies/reaction of the Analyte-2 transcript, and noAnalyte-1 transcript, yielded a combined average hybridization signalfor the IC plus Analyte-1 flasher probes that exceeded only the validitycutoff, and not the Analyte-1 cutoff. This indicated the assay resultswere valid, and established that the test sample was negative forAnalyte-1. These same trials yielded average glower signals thatexceeded the Analyte-2 cutoff, thereby indicating the test sample waspositive for Analyte-2. Finally, trials conducted using 10⁷copies/reaction of the Analyte-1 transcript, and 10⁴ copies/reaction ofthe Analyte-2 transcript yielded a combined average hybridization signalfor the IC plus Analyte-1 flasher probes that exceeded both the validitycutoff and the Analyte-1 cutoff, thereby establishing that the assayresult was valid and that the test sample was positive for Analyte-1.These same trials yielded average glower signals that exceeded theAnalyte-2 cutoff, thereby indicating the test sample was also positivefor Analyte-2. The conclusions presented in Table 4, based on theresults appearing in columns 2 and 3, are consistent with theinterpretations set forth above in Table 2. Of course, conclusionspresented in Table 4 that are relevant to interpretation of the resultsobtained using only the IC and Analyte-1 probes (see column 2) areconsistent with the interpretations set forth above in Table 1.

TABLE 4 Analysis of Experimental Results Avg. IC plus Avg. Analyte-Analyte-1 2 glower flasher signal signal Trial (RLU) (RLU) ConclusionNegative control 210,640 0 Assay valid Analyte-1 (−) Analyte-2 (−)Analyte-1 at 100 c/rxn 2,090,384 0 Assay valid Analyte-2 at 0 c/rxnAnalyte-1 (+) Analyte-2 (−) Analyte-1 at 0 c/rxn 177,431 1,744,838 Assayvalid Analyte-2 at 100 c/rxn Analyte-1 (−) Analyte-2 (+) Analyte-1 at10⁷ c/rxn 2,502,884 1,620,591 Assay valid Analyte-2 at 10⁴ c/rxnAnalyte-1 (+) Analyte-2 (+)

While the present invention has been described and shown in considerabledetail with reference to certain preferred embodiments, those skilled inthe art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe appended claims.

What is claimed is:
 1. A kit for determining the presence or absence ofanalyte polynucleotides in a sample, the kit comprising: an internalcontrol polynucleotide; one or more amplification reagents to co-amplifya first analyte polynucleotide and the internal control polynucleotidein a nucleic acid amplification reaction to produce an internal controlamplicon, and, if the sample contains the first analyte polynucleotide,a first analyte amplicon; and a first hybridization probe having a firstlabel, the first hybridization probe being capable of forming a firstdetectable hybrid with the first analyte amplicon, but incapable offorming a detectable hybrid with the internal control amplicon, and asecond hybridization probe having a second label, the secondhybridization probe being capable of forming a second detectable hybridwith the internal control amplicon, but incapable of forming adetectable hybrid with the first analyte amplicon, wherein the first andsecond labels comprise identical labels.
 2. The kit of claim 1, whereinthe first and second hybridization probes are packaged in a singlecontainer as a combined probe reagent.
 3. The kit of claim 1, whereinthe identical labels are either identical fluorescent labels oridentical chemiluminescent labels.
 4. The kit of claim 1, wherein theamplification reagents to co-amplify the first analyte polynucleotideand the internal control polynucleotide comprise amplification reagentsto co-amplify a second analyte polynucleotide in the nucleic acidamplification reaction to produce a second analyte amplicon.
 5. The kitof claim 4, further comprising a third hybridization probe having athird label, the third hybridization probe being capable of forming athird detectable hybrid with the second analyte amplicon, but incapableof forming a detectable hybrid with the internal control amplicon or thefirst analyte amplicon, wherein the third label is distinguishable fromthe first and second labels.
 6. The kit of claim 1, further comprising anegative calibrator comprising the internal control polynucleotide,wherein the negative calibrator does not comprise the first analytepolynucleotide, and one or more standards, each standard comprising theinternal control polynucleotide and a known amount of the first analytepolynucleotide.
 7. The kit of claim 6, wherein the amplificationreagents to co-amplify the first analyte polynucleotide and the internalcontrol polynucleotide comprise amplification reagents to co-amplify asecond analyte polynucleotide in the nucleic acid amplification reactionto produce a second analyte amplicon.
 8. The kit of claim 7, furthercomprising a third hybridization probe having a third label, the thirdhybridization probe being capable of forming a third detectable hybridwith the second analyte amplicon, but incapable of forming a detectablehybrid with the internal control amplicon or the first analyte amplicon,wherein the third label is distinguishable from the first and secondlabels.
 9. The kit of claim 8, further comprising one or more standardscomprising the internal control polynucleotide and a known amount of thesecond analyte polynucleotide.
 10. The kit of claim 1, wherein theinternal control polynucleotide comprises an RNA transcript.
 11. The kitof claim 1, wherein the amplification reagents comprise a reversetranscriptase and deoxyribonucleotide triphosphates.
 12. The kit ofclaim 11, wherein the internal control polynucleotide comprises an RNAtranscript.
 13. The kit of claim 11, wherein the amplification reagentsfurther comprise ribonucleotide triphosphates, at least onepromoter-primer, and an RNA polymerase.
 14. The kit of claim 1, whereinat least one of the first and second hybridization probes comprises anucleotide analog.
 15. The kit of claim 14, wherein the amplificationreagents comprise a reverse transcriptase, deoxyribonucleotidetriphosphates, ribonucleotide triphosphates, at least onepromoter-primer, and an RNA polymerase.
 16. The kit of claim 1, whereinat least one of the first and second hybridization probes comprises abackbone with a non-nucleotide linker.
 17. The kit of claim 16, furthercomprising a negative calibrator comprising the internal controlpolynucleotide, wherein the negative calibrator does not comprise thefirst analyte polynucleotide, and one or more standards, each standardcomprising the internal control polynucleotide and a known amount of thefirst analyte polynucleotide.
 18. The kit of claim 16, wherein theinternal control polynucleotide comprises an RNA transcript.
 19. The kitof claim 2, wherein the amplification reagents to co-amplify the firstanalyte polynucleotide and the internal control polynucleotide compriseamplification reagents to co-amplify a second analyte polynucleotide inthe nucleic acid amplification reaction to produce a second analyteamplicon.
 20. The kit of claim 2, further comprising a negativecalibrator comprising the internal control polynucleotide, wherein thenegative calibrator does not comprise the first analyte polynucleotide,and one or more standards, each standard comprising the internal controlpolynucleotide and a known amount of the first analyte polynucleotide.21. The kit of claim 2, wherein the amplification reagents comprise areverse transcriptase and deoxyribonucleotide triphosphates.
 22. The kitof claim 2, wherein at least one of the first and second hybridizationprobes comprises a nucleotide analog.
 23. The kit of claim 2, wherein atleast one of the first and second hybridization probes comprises abackbone with a non-nucleotide linker.